EP2428565A1 - Polypeptide having phytase activity and increased temperature resistance of the enzyme activity, and nucleotide sequence coding said polypeptide - Google Patents
Polypeptide having phytase activity and increased temperature resistance of the enzyme activity, and nucleotide sequence coding said polypeptide Download PDFInfo
- Publication number
- EP2428565A1 EP2428565A1 EP11192294A EP11192294A EP2428565A1 EP 2428565 A1 EP2428565 A1 EP 2428565A1 EP 11192294 A EP11192294 A EP 11192294A EP 11192294 A EP11192294 A EP 11192294A EP 2428565 A1 EP2428565 A1 EP 2428565A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- phytase
- sequence
- polypeptide
- dna
- sequences
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
Definitions
- the invention relates to a recombinant DNA molecule which encodes a polypeptide with phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity and the encoded polypeptide as such. More particularly, the invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity, wherein the DNA sequence has been obtained by a variation of wild-type mature E. coli phytase having defined amino acid positions are modified in comparison to the wild-type sequence or N- and / or C-terminal extensions are present. The invention further relates to a method for the expression of recombinant phytase and their use in food and feed technology.
- myoinositol 1,2,3,4,5,6-hexakis dihydrogen phosphate is the major source of inositol and the primary storage form of phosphate in plant seeds.
- myoinositol hexakisphosphate is the major source of inositol and the primary storage form of phosphate in plant seeds.
- about 70% of the phosphate content is present as a mixed potassium, magnesium and calcium salt of phytic acid.
- Seeds, cereals and legumes are important components of food and feed preparations, in particular animal feeds; but also in human food cereals and legumes are becoming increasingly important.
- phosphate units of phytic acid complex complex divalent and trivalent cations such as metal ions, ie nutritionally important ions such as calcium, iron, zinc and magnesium as well as the trace elements manganese, copper and molybdenum.
- phytic acid also binds proteins to some extent through electrostatic interaction.
- Phytic acid and its salts are often not metabolized because they are not absorbed from the gastrointestinal tract, i. neither the phosphorus contained therein nor the chelated metal ions nor the bound proteins are nutritionally available.
- phosphorus is an essential element for the growth of all organisms, food and feed must be supplemented with inorganic phosphate. Very often, the nutritionally essential ions such as iron and calcium must be supplemented. Furthermore, the nutritional value of each diet is reduced because proteins are bound by phytic acid. Consequently, phytic acid is often referred to as an anti-nutritive factor.
- the phosphorus of the phytate is excreted through the gastrointestinal tract of the animals, resulting in undesirable phosphate contamination of the environment, as a result of which, for example, eutrophication of waters and excessive algal growth may occur.
- Phytic acid or phytates can be degraded by phytases.
- Phytases catalyze the hydrolysis of phytate into myo-inositol and / or mono-, di-, tri-, tetra- and / or pentaphosphates as well as inorganic phosphate.
- the 3-phytase preferably hydrolyzes first the ester bond in the 3-position, the 6-phytase prefers first the ester bond in the 6-position.
- Phytic acid-containing plant seeds contain endogenous phytase enzymes. Upon ingestion, the phytates in food or feed are theoretically hydrolyzed by the endogenous plant phytases, by phytases from the intestinal flora and by phytases from the intestinal mucosa. In practice, however, the hydrolysis potential of the endogenous plant phytases and intestinal phytases, if present, is far from sufficient to significantly ensure the bioavailability of the phosphorus bound in the phytates. Therefore, food and feed are often added to exogenous phytases.
- Phytases can be produced by plants as well as by microorganisms. Among the microorganisms, phytase-producing bacteria and phytase-producing fungi and yeasts are known.
- the present invention is therefore based on the object to provide a polypeptide with phytase activity, which has an increased thermal stability or at elevated temperatures has a technologically sufficient activity. Furthermore, the polypeptide with phytase activity should also have increased proteolytic stability.
- the polypeptide should be able to be produced economically.
- the phytase should be more thermostable than the wild-type enzyme.
- the phytase should also retain the essential properties of the natural E. coli wild-type phytase, but be characterized by improved thermal stability.
- the essential properties of the wild type natural phytase include in particular the ability to improve the availability of phosphate in vivo and in vitro by its activity as a phytase as well as a phosphatase, its pH optimum in the acidic with high residual activity in strongly acidic, as well as the suitability as baking aid.
- the polypeptide should be economical and inexpensive to produce.
- expression of the polypeptide in eukaryotic microorganisms should result in a polypeptide with increased thermostability compared to the similarly prepared wild-type phytase.
- phytases from E. coli are described in the literature, e.g. For example, the apA gene coding for a phytase from E. coli K-12 ( Dassa et al., J. Bacteriol. 172: 5497-5500 (1990 )). This gene codes for a periplasmic enzyme that has both acid phosphatase activity and phytase activity (see Greiner et al., Arch. Biochim, Biophys. 303: 107-113 (1993 )). natural Mutants of this enzyme are known (cf. Rodriguez et al., Biochem. Biophys. Res. Comm. 257: 117-123 (1999 )). Genetically modified mutants of the E.
- coli phytase have also been described, leading to increased temperature stability and / or higher specific activity.
- Rodriguez et al. (Arch. Biochem Biophys. 382: 105-112 (2000 )) expressed wild-type AppA and several mutants generated by site-directed mutagenesis in Pichia pastoris to study the effect of N-glycosylation on the temperature stability of the AppA protein. Although glycosylation was not enhanced, a mutant was more active at pH 3.5-5.5 and had more activity after heat treatment than that in P. pastoris produced wild-type protein.
- the patent family based on WO 03/057248 includes the patent applications US 2003/0170293 A1 and US 2003/0157646 A1 , It describes the microbial production of a thermotolerant phytase for animal feed.
- the mutant (Nov9X) of E. coli strain B phytase (appA) is described in E. coli, Pichia pastoris, and Schizosaccharomyces pombe .
- the mutant Nov9X contains 8 amino acid mutations compared to the wild-type enzyme.
- the mutant has improved thermal stability in liquid at 70 ° C compared to the wild-type enzyme.
- the host in which the enzyme is brought to production also has an influence on its thermal stability as described by the same working group in the US patent application US 2003/0157646 A1 is implied.
- the amino acid positional counting is 2 positions higher in NOV9X than in the present invention (W48 NOV9X corresponds to W46 in the present invention).
- the temperature stability or thermostability of proteins depends on a variety of interactions.
- the conformation of proteins is maintained by a large number of weak interactions.
- Stabilization may include all levels of protein structure hierarchy: local packaging of the polypeptide chain, secondary and supersecond structural elements, domains and subunits of a multimeric protein.
- the prior art contains no evidence that a mutation at position 74 (K74D) of the amino acid sequence or a combination of mutations at positions 139 (N139R) and 142 (D142E) or a combination of mutations at positions 145 (L1451) and 198 (L198I) or a mutation at position 200 (V200P) of the wild-type phytase of E. coli or combinations of these mutations lead to improved thermostability of the phytase protein without the beneficial effects and essential properties of the wild-type E. coli phytase. Furthermore, the prior art contains no evidence that the addition of E.
- coli phytase sequence are the constructs PhyM2, PhyM3, PhyM7, PhyM9 and PhyM10. They are shown in Table 1 below. The construction PhyM1 was included as a comparative product.
- polypeptides according to the invention with phytase activity show an increased temperature or thermal stability of their enzyme activity compared with the E. coli wild-type enzyme.
- This improvement in thermal stability can be measured in liquid by differential scanning calorimetry (DSC).
- DSC differential scanning calorimetry
- the improvement in thermostability is evidenced by the greatly increased residual activity of the enzyme mutants over the wild-type enzyme when exposed to the thermal stress used in the pelleting of animal feed.
- the polypeptides of the invention also have an increased stability to proteolytic degradation as it occurs especially in the stomach of poultry and monogastric.
- the phytases according to the invention are particularly suitable for applications in which elevated temperature conditions occur or in which increased proteolytic degradation is to be expected. Examples of use for this purpose are the preparation of the enzyme, in which it must be protected from proteases of the host strain, the production of pelleted animal feed, but also the use as a liquid enzyme in the post-pelleting application to the pelleted animal feed.
- the phytase sequence according to the invention preferably contains at least one and particularly preferably at least two of the abovementioned variations. Any combination of the above-mentioned mutation variants are possible in order to adapt the thermostability of the phytase to the prevailing conditions of the particular application.
- a preferred combination option is the combination of i), ii) and iv), which is referred to below as the genotype PhyM10.
- an N-terminal or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. Awamori may be present. Even the sole presence of these sequence sections already leads to an increase in the thermal stability.
- the N-terminal portion of the A. niger var. Awamori acid phosphatase is the first 51 amino acids of the mature protein (FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS). The last 4 amino acids of this polypeptide replace the first 4 amino acids (QSEP) of mature E.
- the N-terminal part or parts thereof, the first 40 amino acids of the mature protein of Aspergillus niger phytase can be used (SCDTVDQGYQ CFSETSHLWG QYAPFFSLAN ESVISPEV-PA).
- SCDTVDQGYQ CFSETSHLWG QYAPFFSLAN ESVISPEV-PA The appendix to the E. coli phytase may also occur elsewhere than at position 4 described as long as the activity of the enzyme is maintained.
- the C-terminal extension, the last 29 amino acids (LSFWWNYNTT TELNYRSSPI ACQEGDAMD) of the acid phosphatase from A. niger var were.
- thermostability of the phytase according to the invention can be further increased in commercial products, for. B. by adding substances into the ultrafiltration concentrate before drying or granulation.
- a stable formulation of the phytase enzyme of the present invention can be prepared by spraying a mixture of a liquid enzyme solution onto a filler such as maltodextrin and then drying the mixture, or 20% -60% skimmed milk powder (w / o) before drying the liquid enzyme solution.
- w based on total protein of the enzyme solution and / or 1% -5% calcium propionate (w / w based on total protein of the enzyme solution) was added and the pH was adjusted to a defined value, in particular to pH 5.2 ⁇ 0.5.
- the reduction of moisture and the binding interactions of the phytase with the filler protect the enzyme, in addition to its stability defined in its structure, from environmental influences such as temperature extremes that may occur during the manufacture of the feed. Dry and liquid formulations can be further stabilized by reducing the activity of potential proteolytic enzymes which may be present as by-products in the liquid fermentation mixture used to prepare the enzyme of the present invention.
- the DNA sequence corresponding to the phytase sequence mutated according to the invention can be realized using any codon usages.
- the codon usage of the microorganism used for expression can be used, but it can also be used the E. coli codon usage or a variant thereof.
- the mutated E. coli phytase sequence according to the invention may contain further sequence variations. Any variations may be made in addition to the mutations described above as long as the property of increased temperature stability is not adversely affected and as long as the enzymatic activity and other essential properties of the E. coli wild type phytase are maintained.
- addition and / or deletion molecules of the polypeptide modified according to the invention can be used.
- the modified polypeptide with phytase activity according to the invention can be extended by adding further sequences at the N-terminal and / or C-terminal end.
- hybrid molecules can be produced which have further advantageous properties.
- fusion proteins or native highly secreted proteins can be added, thereby improving secretion efficiency.
- a part of the CBH2 protein from Trichoderma reesei, from amino acid Met 1 to Ser 86, is preferably used for this purpose.
- Sequence sections of the polypeptide with phytase activity can also be deleted according to the invention as long as the property of increased temperature stability is not impaired while maintaining the phytase activity.
- the mutations, elongations and truncations can be carried out in a manner known per se according to methods well known in the art.
- modifications of the polypeptide having phytase activity contemplated above correspond to corresponding mutations or modifications of the associated DNA molecule.
- Contemplated sequences that hybridize under relaxed or stringent conditions with the sequences of the invention. Stringent conditions are: hybridization at 65 ° C, 18 h in dextran sulfate solution (GenescreenPlus, DuPont), then wash the filter for 30 min each first with 6 x SSC, twice 2 x SSC, twice 2 x SSC with 0, 1% SDS followed by 0.2 x SSC at 65 ° C (membrane transfer and detection methods, Amersham).
- the invention also relates to those sequences which have a homology of at least 70%, more preferably at least 80%, even more preferably at least 90% and in particular at least 95% with the claimed nucleotide sequence or the claimed parts thereof, as long as the respective sequences for increasing the Temperature stability of the encoded by them polypeptide with phytase activity.
- the homology 70 is up to 100 percent.
- Homology is defined as the degree of identity. The degree of identity is thereby preferably determined by determining the number of residues of the shorter sequence involved in the comparison and having a "corresponding" counterpart in the other sequence.
- the homology is preferably determined in a conventional manner using the usual algorithms.
- cDNAs of the respective mature proteins are used for comparison.
- sequence counterparts are determined according to the invention with the aid of known computer programs.
- An example of such a program is the program Clone Manager Suite, which contains the program part Align Plus and is distributed by Scientific & Educational Software, Durham, NC, USA.
- a comparison of two DNA sequences as defined above is carried out under the option of local alignment either by the method FastScan - MaxScore or by the method Needleman-Wunsch while maintaining the default values.
- the program version "Clone Manager 7 Align Plus 5" with the functions "Compare Two Sequences / Local Fast Scanning Max Score / Compare DNA sequences” was used to calculate the homology.
- the algorithms available from the following sources were used: Hirschberg, DS (1975) A linear space algorithm for computing longest common subsequences, Commun Assoc Comput Mach 18: 341-343 ; Myers, EW and W. Miller. (1988) Optimal alignments in linear space, CABIOS 4: 1, 11-17 ; Chao, KM, WR Pearson and W. Miller. (1992) Aligning two sequences within a specified diagonal band, CABIOS 8: 5, 481-487 ,
- the invention further relates to DNA sequences which are related due to the degeneracy of the genetic code with the sequences of the invention as well as allelic variants thereof.
- the degeneracy of the genetic code may be due to natural degeneration or due to a specially chosen codon usage.
- Naturally occurring allelic variants may be synthesized using well-known molecular biology techniques, such as. The polymerase chain reaction (PCR) and hybridization techniques.
- a DNA sequence encoding a polypeptide of the invention can be used to transform any host cells, such as fungi, yeast, bacteria, plants or mammalian cells. Such transformed cells are characterized by a production and ev. Secretion of phytase with increased thermostability. The thus produced phytase enzyme with increased thermostability also leads to an efficient phosphate release from phytates.
- a polypeptide or enzyme with phytase activity or a phytase is intended to refer to any enzyme capable of causing the release of inorganic phosphate from various myo-inositol phosphates.
- myoinositol phosphate (phytase) substrates are phytic acid and any salts thereof, for example sodium phytate or potassium phytate or mixed salts. Any positional isomers of the di-, tri-, tetra- or pentaphosphates of myo-inositol can serve as a phytase substrate.
- a phytase variant of the invention comprises polypeptide variants derived from a particular phytase by deletion or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein, deletion or addition of one or more amino acids at one or more sites in derived from the native protein or substitution of one or more amino acids at one or more sites in the phytase.
- the generation of such variants is generally known in the art.
- amino acid sequence variants of the polypeptides can be made by mutation in the DNA.
- the invention also relates to isolated or substantially purified nucleic acid or protein compositions.
- An isolated and purified polynucleotide / polypeptide or segment thereof refers to a polynucleotide or polypeptide or segment thereof that is isolated from its native environment.
- An isolated polynucleic acid segment or polypeptide may be in purified form or may be in a non-native environment, such as in a transgenic host cell.
- an isolated or purified polynucleotide segment or protein or biologically active portion thereof is substantially free of further cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemical compounds.
- an isolated polynucleotide is free of sequences (preferably protein coding sequences) that naturally contain the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived flank.
- the isolated nucleic acid molecule may comprise less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb nucleotide sequences that naturally comprise the nucleotonic acid molecule in the genomic DNA of the cell flanking the nucleic acid derived.
- a protein that is substantially free of cellular material includes protein or polypeptide compositions having less than about 70%, 50%, 30%, 20%, 10%, 5% (by dry weight) of contaminating protein.
- the culture medium comprises less than about 70%, 50%, 30%, 20%, 10% or 5% (by dry weight) of the chemical precursors or non-proteinaceous chemicals substances.
- Fragments and variants of the nucleotide sequences of the invention or proteins or protein segments encoded thereby are also included in the invention. Fragment refers to part of the nucleotide sequence or part of the amino acid sequence and therefore a portion of the polypeptide or protein encoded thereby.
- the invention also relates to expression cassettes which can be used to introduce the phytase-encoding open reading frame into a host cell. They preferably comprise a transcription initiation region linked to the open reading frame. Such an expression cassette may have a plurality of restriction sites for insertion of the open reading frame and / or other DNAs, e.g. A transcriptional regulatory region and / or selectable marker genes.
- the transcriptional cassette in the 5 ' ⁇ 3' direction of transcription comprises a transcriptional and translational initiation region, the DNA sequence of interest, and a transcriptional and translational stop region that is functional in a microbial cell.
- the termination region may be native to the transcription initiation region, may be native to the DNA sequence of interest, or may be derived from any other source.
- ORF open reading frame
- “Functional linkage” in the context of a nucleic acid refers to a compound as part of the same nucleic acid molecule in proper positioning and orientation for the transcription initiation of the promoter.
- DNA operably linked to a promoter is under the transcriptional initiation regulation of the promoter.
- Coding sequences may be operably linked to the regulatory sequence in sense or antisense orientation.
- polypeptides functional means Link the compound as part of the same polypeptide, ie via peptidyl bonds.
- Promoter usually refers to the nucleotide sequence upstream (5 ') to the coding sequence and controls the expression of the coding sequence by providing recognition for the RNA polymerase and other factors required for proper transcription.
- the promoter used in the invention may comprise a minimal promoter, i. a short DNA sequence from a TATA box and other sequences specifying the transcription start site to which the expression control elements are attached.
- the promoter of the invention may also comprise a nucleotide sequence comprising a minimal promoter and regulatory elements capable of controlling the expression of a coding sequence or functional RNA.
- This type of promoter sequence consists of proximal and distal upstream elements, the latter elements often being referred to as enhancers.
- an enhancer is a DNA sequence that can stimulate promoter activity and may be an element inherent in the promoter or an inserted heterologous element to enhance the level of expression or tissue specificity of a promoter. It can function in both orientations and can function even on upstream or downstream placement of the promoter. Both enhancers and other upstream promoter elements sequence-specifically bind DNA-binding proteins that mediate their effects.
- Promoters may be derived in their entirety from a native gene, or may be composed of various elements derived from various naturally occurring promoters, or may even be composed of synthetic DNA segments.
- a promoter may also contain DNA sequences that participate in the binding of protein factors which control the efficiency of transcription initiation in response to physiological or developmental conditions.
- TATA elements which are inactive or have greatly reduced promoter activity in the absence of upstream activation are termed minimal promoters or core promoters.
- a minimal or core promoter thus consists only of all basic elements necessary for transcription initiation, e.g. As a TATA box and / or an initiator.
- the invention also relates to the DNA-containing vectors of the invention.
- vectors include any of plasmids, cosmids, phage and other vectors in double-stranded or single-stranded, linear or circular form which may optionally be self-transmissive or mobilizable and which may transform a prokaryotic or eukaryotic host either integrally into the cellular genome or which are extra-chromosomally present (eg, autonomously replicating plasmids with a replication origin).
- Vectors, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and DNA segments for use in transforming cells generally include the phytase-encoding DNA of the present invention, as well as other DNA such as cDNA, a gene, or genes found in the cells are to be introduced. These DNA constructs may include other structures such as promoters, enhancers, polylinkers or regulatory genes, as necessary.
- One of the DNA segments or genes selected for cellular delivery suitably encodes / encodes a protein that is expressed in the transformed (recombinant) cells thus obtained, resulting in a screenable or selectable Feature and / or confer an improved phenotype on the transformed cell.
- the construction of vectors which can be used in accordance with the invention is known to a person skilled in the art in light of the above disclosure (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed., Coldspring Harbor Laboratory Press, Plainview, NY (1989) )).
- the expression cassette of the invention may contain one or more restriction sites to place the phytase-encoding nucleotide under the regulation of a regulatory sequence.
- the expression cassette may also contain a termination signal operably linked to the polynucleotide as well as regulatory sequences needed for proper translation of the polynucleotide.
- the expression cassette containing the polynucleotide according to the invention may be chimeric, ie at least one of its components is heterologous with respect to at least one of the other components.
- the expression of the polynucleotide in the expression cassette may be under the control of a constitutive promoter, an inducible promoter, a regulated promoter, a viral promoter or a synthetic promoter.
- the vectors may already contain regulatory elements, e.g. Promoters, or the DNA sequences of the invention can be manipulated to contain such elements.
- Suitable promoter elements which can be used are known in the art and are for example Trichoderma reesei the cbh 1 or cbh 2 promoter, for Aspergillus ory zae the amy promoter, for Aspergillus niger the xyl, glaA-, alcA, aphA, tpiA, gpdA, sucl and pkiA promoters.
- Suitable promoter elements that can be used for expression in yeast are known in the art and are, for example, the pho5 promoter or the gap promoter for expression in Saccharomyces cerevisiae and for Pichia pastoris, e.g. B. aoxl promoter or the fmd promoter or the mox promoter for H. polymorpha.
- DNA suitable for introduction into cells may also comprise DNA derived from or isolated from any source.
- An example of a deduced DNA is a DNA sequence which has been identified as a useful fragment in a given organism and which has then been chemically synthesized in substantially pure form.
- An example of such a DNA is a suitable DNA sequence obtained, for example, using restriction endonucleases so that it can be further manipulated according to the invention, for example amplified.
- DNA is commonly referred to as recombinant DNA.
- a suitable DNA comprises wholly synthetic DNA, semisynthetic DNA, DNA isolated from biological sources, and DNA derived from in-blood RNA.
- the introduced DNA is not an original part of the genotype of the recipient DNA, but according to the invention a gene from a given genotype can also be isolated and eventually altered and subsequently multiple copies of the gene can be introduced into the same genotype, e.g. To enhance production of a given gene product.
- the introduced DNA includes, without limitation, DNA from genes such as bacteria, yeasts, fungi or viruses.
- the introduced DNA may comprise modified or synthetic genes, parts of genes or chimeric genes, including genes from the same or a different genotype.
- the DNA used for the transformation according to the invention may be circular or linear, double-stranded or single-stranded.
- the DNA is in the form of a chimeric DNA, such as plasmid DNA, which also contains coding regions flanked by regulatory sequences that aid in the expression of the recombinant DNA present in the transformed cell.
- the DNA itself may contain or consist of a promoter that is active in a cell that is from a source other than the cell is differentiated, derived or it can be used a promoter that is already present in the cell, ie the transformation target cell.
- the introduced DNA is relatively small, less than about 30 kb, to minimize sensitivity to physical, chemical or enzymatic degradation, which increases with the size of the DNA.
- Yeast or fungal expression vectors may include a replication origin, a suitable promoter and enhancer, and also include any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed 5 'flanking sequences.
- suitable host cells are: fungal cells of the genus Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium, etc., such as yeasts of the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula, Pichia and the like.
- Suitable host systems are, for example, fungi such as Aspergilli, z. Aspergillus niger (ATCC 9142) or Aspergillus ficuum (NRLL 3135) or Trichoderma (eg Trichoderma reseei QM6a) and yeasts such as Saccharomyces, e.g. B.
- Saccharomyces cerevisiae or Pichia such as. B. Pichia pastoris or Hansenula, z. B. H. polymorpha (DSMZ 70277).
- Such microorganisms may be used by recognized depositories, e.g. For example, the American Type Culture Collection (ATCC), the Centraalbureau voor Schimmelcultures (CBS) or the German Collection for Microorganisms and Cell Cultures GmbH (DSMZ) or any other depositories.
- ATCC American Type Culture Collection
- CBS Centraalbureau voor Schimmelcultures
- DSMZ German Collection for Microorganisms and Cell Cultures GmbH
- the expression cassette may contain, in the 5'-3 'direction of transcription, a transcriptional and translational initiation region of the polynucleotide of the present invention and a transcription and termination region that is functional in vivo or in vitro.
- the termination region may be native to the transcription initiation region or may be native or polynucleotide related be of another origin.
- the regulatory sequences may be located upstream (5 'noncoding sequences), within (intron) or downstream (3' noncoding sequences) of a coding sequence, and affect transcription, RNA processing or stability and / or translation of the associated coding sequence.
- Regulator sequences may include, without limitation, enhancers, promoters, repressor binding sites, translational leader sequences, introns, or polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences that are a combination of synthetic and natural sequences.
- the vector used in the invention may also comprise suitable sequences for amplification of expression.
- promoters which can be used in the present invention are promoters known to control expression in the eukaryotic cells. Any promoters capable of expression in filamentous fungi can be used. Examples are a promoter which is strongly induced by starch or cellulose, e.g. A promoter for glucoamylase or ⁇ -amylase from the genus Aspergillus or cellulase (cellobiohydrolase) from the genus Trichoderma, a promoter for enzymes in the glycolytic pathway, such as phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPD), etc.
- Preferred is the cellobiohydrolase I, the cellobiohydrolase II, the amylase, the glucoamylase, the xylanase or the enolase promoter.
- Overexpression can be achieved by insertion of one or more extra copies of the selected gene.
- antisense downregulation and “sense downregulation”. In general, these methods are referred to as gene silencing. Both of these methods lead to an inhibition of the expression of the target gene.
- introns have been shown to have the potential to enhance transgene expression.
- the expression cassette may include other elements, for example, those that may be regulated by endogenous or exogenous elements, such as zinc finger proteins, including naturally occurring zinc finger proteins or zinc protein chimeric proteins.
- the expression cassette used in the invention may further contain enhancer elements or upstream promoter elements.
- Vectors for use in the invention can be engineered to contain an enhancer element.
- the constructs of the invention thus comprise the gene of interest together with a 3 'DNA sequence which acts as a signal to terminate transcription and allow polyadenylation of the mRNA thus obtained.
- Any signal sequences that allow secretion from the chosen host organism can be used.
- Preferred signal sequence is the phytase signal sequence from Aspergillus niger or derived therefrom signal sequences for secretion from filamentous fungi.
- leader sequences can also be used since the DNA sequence between the transcription start site and the start of the coding sequence, ie the untranslated leader sequence, can influence gene expression.
- Preferred leader sequences include sequences that direct the optimal expression of the attached gene, ie, they include a preferred consensus leader sequence that increases or maintains mRNA stability and prevents inappropriate translation initiation. The choice of such sequences is well known to one skilled in the art.
- a selectable or screenable marker gene can be incorporated into the expression cassette.
- marker genes are well known to one skilled in the art.
- the expression cassette or a vector construct containing the expression cassette is introduced into a host cell.
- a variety of techniques are available and well known to those skilled in the art for introducing constructs into a host cell.
- the transformation of microbial cells can be carried out using polyethylene glycol, calcium chloride, viral infection, DEAE-dextran, phage infections, electroporation and other methods known in the art.
- the transformation of mushrooms can after Penttilä et al., Gene 61: 155-164, 1987 be performed.
- the introduction of a recombinant vector into yeasts can be carried out by methods known per se, including electroporation, use of spheroplasts, lithium acetate and the like.
- the expression cassette or DNA sequence of the present invention can be inserted into vectors by methods known per se in order to overexpress the encoded polypeptide in suitable host systems.
- DNA sequences as such can also be used to transform suitable host systems of the invention to achieve overexpression of the encoded polypeptide.
- the encoded phytase may be secreted either from the medium if the phytase is secreted into the medium or from the host organism if the phytase is intracellularly e.g. B. is present in the periplasmic space, concentrated and / or isolated according to known methods.
- Known method of separating the insoluble Culture medium and biomass components followed by phytase concentration techniques may be used to prepare concentrated phytase solutions or to prepare the phytase to dry.
- filtration methods or centrifugation methods may be used to separate the insoluble components, followed by ultrafiltration methods for concentration, or cross-flow filtration methods are used. Drying may be by freeze and spray drying, granulation, extrusion or other methods.
- Known methods of protein purification can be used to isolate the phytases of the invention. For example, various chromatographic or gel chromatographic methods can be used singly or in combination.
- the enzyme of the invention may or may not be covalently modified by glycosylation. In eukaryotic cells, glycosylation of the secreted proteins serves to modulate protein folding, conformational stability, thermal stability, and resistance to proteolysis.
- a glycosylated variant of the enzyme may be preferred over a non-glycosylated variant.
- a glycosylated phytase in animal feed to protect the enzyme from thermal denaturation during food pelleting and proteolytic inactivation upon passage through the animal's stomach promotes distribution of the active enzyme in the intestinal tract and to the site of action.
- a thermolabile, ie, non-glycosylated, and more susceptible to protolytic degradation may be preferred.
- the invention also relates to phytase compositions containing the polypeptide of the invention.
- phytase compositions are liquid or dry.
- Liquid compositions preferably contain the phytase enzyme in a purified or enriched form. however also, adjuvants such as a stabilizer such as glycerol, sorbitol or monopropylene glycol, additives such as salts, sugars, preservatives, pH adjusters, proteins and phytate or salts of myoinositol phosphates (a phytase substrate) may be added.
- Typical liquid compositions are aqueous or oily slurries. The liquid compositions may be added to a food or feed before or after a possible pelleting or processing step.
- Dry compositions may be freeze-dried, spray-dried, granulated or extruded compositions which may contain only the enzyme. However, before drying, substances may also be added to control the pH, as well as other additives and fillers such as maltodextrins, lactose, skimmed milk powder, salts of divalent cations such as Mg, Ca, Zn, etc. Dry compositions may be granules which may be easily mixed with, for example, food or feed components, or more preferably form a component of a premix. The particle size of the enzyme granules is preferably compatible with that of the other components of the mixture. This allows safe and convenient means to incorporate enzymes into, for example, processed foods, premixes or animal feed.
- a stable formulation of the phytase enzyme of the present invention can be prepared by spraying a mixture of a liquid enzyme solution onto a filler such as maltodextrin and then drying the mixture, or 20% -60% skimmed milk powder (w / o) before drying the liquid enzyme solution.
- w based on total protein of the enzyme solution and / or 1% -5% calcium propionate (w / w based on total protein of the enzyme solution) was added and the pH was adjusted to a defined value, in particular to pH 5.2 ⁇ 0.5.
- the reduction in moisture and the binding interactions of the phytase with the filler protect the enzyme, in addition to its stability defined in its structure, from environmental influences such as temperature extremes that occur during the manufacture of the feed may occur.
- Dry and liquid formulations can be further stabilized by reducing the activity of potential proteolytic enzymes which may be present as by-products in the liquid fermentation mixture used to prepare the enzyme of the present invention.
- the resulting dry enzyme mixture can be used as a feed supplement for use in poultry and pig breeding.
- a reduction in phosphate supplementation leads to a decrease in phosphate contamination, which in turn significantly reduces the environmental impact of intensive stock farming.
- an agglomeration granulate can be produced in further steps.
- a high shear mixer is used which coagglomerates filler and the enzyme and granules are formed.
- Absorption granules are prepared by coating cores from a carrier material by the enzyme according to the invention.
- Typical fillers are salts, such as disodium sulfate.
- Other fillers include kaolin, talc, magnesium aluminum silicate, and cellulosic fibers.
- binders such as dextrins are also incorporated in the agglomeration granules.
- Typical support materials include starch, e.g. B. in the form of cassava, potato and cereals, especially corn, rice and wheat, or proteinaceous products such. B. soy protein. Salts can also be used.
- the granules are optionally coated with a coating mixture.
- a coating mixture comprises coating agents, preferably hydrophobic coating agents, dehydrated palm oil and talc, and optionally other additives such as calcium carbonate or kaolin in order to improve the bioavailability at the intended site.
- mixtures with phytase may contain other substances such as coloring agents, aroma compounds, stabilizers, vitamins, minerals, other food or feed enzymes and the like. This is especially true for the so-called premixes.
- a food or feed additive is a substantially pure compound or compound of a number of compounds which is intended or suitable to be added to food or feed.
- it is a substance that becomes a component of a food or feed according to its purpose or influences properties of a food or feed product.
- a phytase additive is intended to refer to a phytase which is not a natural constituent of the substances principally used for food and feed or is absent in their natural concentration therein.
- the phytase is added to the feed separately from the feeds alone or in combination with other feed additives.
- a typical premix will usually comprise one or more compounds such as vitamins, minerals or feed-enhancing enzymes, and suitable carriers and / or excipients.
- a ready-to-use phytase additive is an additive that is not produced in situ in animal feed or in processed foods.
- a ready-to-use phytase additive may be administered to humans or animals directly or preferably directly after mixing with other ingredients of the feed or foods.
- a feed additive according to this aspect of the present invention is combined with other feeds and additives to obtain a premix or supplemental feed.
- Such other feed ingredients include one or more other (preferably thermostable) enzyme supplements, other feed additives, mineral feeds and amino acids.
- the (combined) feed additives thus obtained may comprise several different types of compounds and may then be mixed in their appropriate amount with feeds such as cereal and protein carriers to form a composite animal feed.
- the processing of these components into an animal feed may be known per se after mixing Processing devices such as a Doppelpelletiermaschine, a Dampfpelletierers, an expander or an extruder are performed.
- a food additive according to this embodiment of the present invention may be combined with other food components to produce processed food products.
- Such other food components include one or more enzyme supplements, vitamins, minerals and trace elements.
- the combined dietary supplement thus obtained may then be mixed in an appropriate amount with other food components, such as cereals and vegetable proteins, to yield a processed food.
- the processing of these components into a processed foodstuff may be carried out using processing devices known per se.
- the phytase compositions of the invention additionally comprise an effective amount of one or more enzymes for food or feed, preferably selected from alpha-galactosidases, beta-galactosidases, laccases, other phytases, phosphatases, endoglucanases, in particular endo-beta-1,4 Glucanases, endo-beta-1,3 (4) -glucanases, endo-1,2-beta-glucanases and endo-1,3-alpha-glucanases, cellulases, xylosidases, galactanases, especially arabinogalactan-endo-1 , 4-beta-galactosidases and arabinogalactan-endo-1,3-beta-galactosidases, pectin degrading enzymes, in particular pectinases, pectin esterases, pectin lyases, pectin
- the animal feed additive according to the invention is administered to the animal before or simultaneously with the feed.
- the animal feed additive according to the invention is preferably administered to the animal simultaneously with the feed.
- An effective amount of phytase in food or feed is about 10-20,000 PPU / kg, preferably about 10-15,000 PPU / kg, more preferably about 10-10,000 PPU / kg, even more preferably about 50-5,000 PPU / kg, especially 50-2,000 PPU / kg feed or food.
- the invention also relates to the use of phytase for the processing and production of food and feed.
- Food grains and flours can be enzymatically treated with phytase to reduce the phytin content of the raw materials. Reduced phytin levels improve food quality by increasing the availability of essential minerals such as iron, calcium and zinc.
- the use of phytase during processing can improve the overall efficiency of food production. For example, the addition of phytase to white soybean flakes during the production of a soy protein isolate can significantly increase the yield and quality of the extractable protein.
- the phytase is only active during production and processing and is no longer active in the final product.
- animal feed components such as toasted soybean meal or canola meal with phytase may be pretreated prior to the actual manufacturing process.
- the removal of anti-nutritive factors in animal feed components prior to preparation results in a higher physiological quality and more valuable animal feed ingredients.
- the phytase is active during production and is generally no longer active in the digestive tract of the animal after ingestion of the treated feed.
- the present invention relates to the use of the phytase of the invention as a digestive aid.
- Phytase in tablet form can be taken with food intake to distribute the active enzyme in the gastrointestinal tract.
- the phytase according to the invention can also be advantageously used in monogastric and polygastric animals, in particular in young calves. Feed for fish and shellfish may also be supplemented with phytase to improve feed utilization and reduce levels of excreted phosphorus in intensive livestock.
- the feed of the invention may also animals such as poultry, z. As chickens, turkeys, geese, ducks and pigs, horses, cattle, sheep, goats, dogs and cats and fish and shellfish are administered. However, it is particularly preferred that the feed according to the invention is administered to pigs or poultry.
- Phytase formulations according to the invention can also be combined with other ingredients, thereby creating new and particularly advantageous feed compositions.
- inorganic phosphate is added to the feed to ensure adequate phosphorus supply to the animals.
- these feeds contain too much total phosphate, leading to pollution of the environment with phosphate.
- the animal feed of the present invention comprises the combination of a phytase of the invention with animal feed ingredients to yield a feed containing substantially reduced levels of added inorganic phosphorus.
- the feed according to the invention comprises typical feed ingredients, micronutrients, trace elements, vitamins, etc.
- phytase and inorganic phosphorus are between 50 and 20,000 units Phytase / kg feed and less than 0.45% inorganic phosphorus, preferably between levels of 100-10,000 units phytase / kg feed and less than 0,225% inorganic phosphorus, more preferred levels of 150-10,000 units phytase / kg feed and less than 0, 15% inorganic phosphorus, more preferably levels of 200-20,000 units phytase / kg feed and no additional addition of inorganic phosphorus.
- the invention also relates to methods for improving weight gain and feed conversion ratio (FCR) in animal nutrition and to the use of the phytases according to the invention in these methods.
- a phytase of the invention allows for improved weight gain and improved feed utilization, especially in the context of feed containing little inorganic phosphate.
- the content of inorganic phosphate in feed can be reduced below levels of 0.45%, preferably below 0.225%.
- no inorganic phosphate is added.
- Increased availability of phosphate as a result of the addition of the enzyme of the invention the bone mineralization of the animals can be significantly improved, which is particularly important in fast-growing animals of great importance.
- the invention relates to the use of the enzyme according to the invention in baking, whereby the development, elasticity and / or stability of the dough and / or the volume, the crumb structure and / or the resistance of the baked product to staling is improved.
- the enzyme preparation according to the invention for the preparation of dough or baked products from any type of flour, z. B. based on rye, barley, oats or corn can be used, the enzyme preparation according to the invention proved to be particularly useful in the preparation of doughs or bakery products from wheat or from a substantial proportion of wheat.
- the bakery products that can be made with an enzyme preparation of the invention include bread, rolls, Baguette and the like.
- the enzyme preparation of the invention with a further enzyme activity such as.
- Phytase activity was measured in an assay mixture of 0.5% phytic acid (about 5 mM), 200 mM sodium citrate, pH 5.0. After 15 minutes incubation at 37 ° C, the reaction was stopped by adding an equal volume of 15% trichloroacetic acid.
- the liberated phosphate ions were prepared by mixing 100 ⁇ l of the assay mixture with 900 ⁇ l of H 2 O and 1 ml of 0.6 MH 2 SO 4 , 2% ascorbic acid and 0.5% ammonium molybdate after incubation at 50 ° C. and a duration of 20 min, quantified at 820 nm. Potassium phosphate standard solutions were used as reference.
- the plasmid pET-PhyM2 contains the E. coli phytase sequence (Dassa et al., 1990, Accession No. M58740) with the changes of amino acids N139R and D142E.
- the DNA sequence comprising an open reading frame of 1230 bp with the CAG (Gln) codon at position 1 and encoding a 410 amino acid enzyme was prepared using the codon usage of T. reesei (http: //www.kazusa .or.jp / codon) into the plasmid pET26b (+) (Novagen, Germany, modified by Brain, Germany).
- a PCR-based oligonucleotide-directed method was used for the realization of mutagenesis.
- the plasmid is in Fig. 1 and was filed under the accession number DSM 18715 on 18.10.2006.
- the PhyM2 gene coding for E. coli phytase was amplified from the plasmid pET-PhyM2 by PCR.
- the PCR product was cut with the restriction enzymes SpeI and PacI and inserted into the Spel and PacI sites after the T. reesei cbhlI gene fragment in plasmid pAB490. This gave rise to an open reading frame encoding the CBHII-KexII-PhyM2 fusion.
- the resulting plasmid is called pAB490-PhyM2 ( Fig. 2 ) and was mapped by restriction endonucleases. The phytase sequence was confirmed by sequencing.
- the plasmid pAB490 is based on pUC18 and contains the cbhll gene fragment ( Nucleotides 1-307 correspond to amino acids M1 to S86, Teeri et al, 1987, Gene 51: 43-52 Accession No. 16190) under the control of the cbhI promoter and cbhI terminator from the plasmid pALK487 ( WO 94/28117 ).
- the E. coli phytase sequence containing the mutation of amino acids N139R and D142E is fused at the 3 'end of the cbhll gene fragment via a KexII site.
- the Kex sequence contains the following amino acids: RTLVKR.
- cbhl Terminator The 0.75 kb BamHI / Stul fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
- amdS gene The gene including its promoter and its terminator was isolated from Aspergillus nidulans VH1-TRSX6 and codes for acetamidase ( Hynes et al., 1983, Mol. Cell. Biol. 3: 1430-1439 ; Kelly and Hynes, 1985, EMBO J. 4: 475-479 ). Acetamidase allows the strain to grow using acetamide as the sole nitrogen source and this characteristic was used to select the transformants.
- cbhl 3 'fragment The fragment (1.7 kb, BamHI / EcoRI, starting 1.4 kb after the stop codon of the gene) was isolated from T. reesei ALKO2466.
- the strain ALKO2466 is derived from strain ALKO233 ( Harkki et al., 1991, Enzyme Microb. Technol. 13: 227-233 ).
- the 3 'fragment, along with the promoter region, is used to target the phytase expression cassette into the cbhl locus by homologous recombination.
- the sequence of plasmid pAB490-PhyM2 was confirmed by restriction enzyme mapping and sequencing.
- the plasmid pET-PhyM7 contains the E. coli phytase (Dassa et al., 1990, Accession No. M58740) sequence using the codon usage of T. reesei (http://www.kazusa.or.jp/codon). with the mutation K74D.
- the construction and the cloning of the plasmid pET-PhyM7 was carried out analogously to the preparation of the plasmid pET-PhyM2 described in Example 2.
- the primers used to insert the amino acid position 74 mutations are: cggactcctggctgacaagggatgcccgc (SEQ ID NO: 5) gcgggcatcccttgtcagccaggagtccg (SEQ ID NO: 6)
- the plasmid pET-PhyM7_ has been deposited under the accession number DSM 18716 on 18.10.2006.
- the construction and the cloning of the plasmid pAB490-PhyM7 was carried out analogously to the preparation of the plasmid pAB490-PhyM2 described in Example 2.
- the sequence of the plasmid pAB490-PhyM7 was determined by sequencing approved.
- the expression cassette isolated from the plasmid pAB490-PhyM7 therefore contains the same elements as described in Example 2, with the exception of the new specific phytase sequence of the genotype PhyM7.
- the phytase variant PhyM3 contains the E. coli phytase (Dassa et al., 1990, Accession No. M58740) sequence using the codon usage of T. reesei (http://www.kazusa.or.jp/codon) with the mutation V200P.
- the DNA sequence with the CAG (Gln) codon in position 1 comprises an open reading frame of 1230 bp and encodes a 410 amino acid enzyme.
- the 18 amino acid long signal peptide of A's phytase. niger was used to detect the phytase mutant of E. coli from Trichoderma reesei .
- the mutation V200P by the PCR method analogous to the principle of Tomic et al. (1990, Nucleic Acids Research, 18 (6), 1656 ) and Vallette et al. (1989, Nucleic Acids Research, 17 (2), 723-733 ) carried out.
- the PCR product was cut with Avrll and Pacl and inserted into the SpeI and PacI cleavage sites after the T. reesei cbh1 promoter in the plasmid pAB489.
- plasmid pAB489-PhyM3 is the gene of E.
- the modified signal peptide of A. niger- phytase was used to secrete the E. coli phytase from Trichoderma reesei .
- the E. coli phytase with the mutation V200P sequence including the A. niger phytase signal sequence was inserted between the cbhl promoter and the cbhl terminator.
- cbhI terminator The 0.75 kb BamHI / StuI fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
- amdS gene The gene including its promoter and its terminator was isolated from Aspergillus nidulans VH1-TRSX6 and codes for acetamidase ( Hynes et al., 1983, Mol. Cell. Biol. 3: 1430-1439 ; Kelly and Hynes, 1985, EMBO J. 4: 475-479 ). Acetamidase allows the strain to grow using acetamide as the sole nitrogen source and this characteristic was used to select the transformants.
- cbhl 3 'fragment The fragment (1.7 kb, BamHI / EcoRI, starting 1.4 kb after the stop codon of the gene) was isolated from T. reesei ALKO2466.
- the strain ALKO2466 is derived from strain ALKO233 ( Harkki et al., 1991, Enzyme Microb. Technol. 13: 227-233 ).
- the 3 'fragment was used together with the promoter region to properly integrate the phytase expression cassette into the cbh1 locus by homologous recombination.
- the phytase variant PhyM3 gene was cloned into the plasmid pAB2004.
- the resulting plasmid has the name pUC-PhyM3 ( Fig. 3 ) and deposited as DSM 18717 according to the above conditions.
- the plasmid pAB2004 is based on the plasmid pUC18 and was prepared by inserting additional sites into the HindIII-EcoRI sites.
- the phytase variant PhyM9 contains the E. coli phytase (Dassa et al., 1990, Accession No. M58740) Sequence as a synthetic gene using the codon usage of T. reesei (http://www.kazusa.or.jp/codon) with mutations L1451 and L1981.
- the DNA sequence with the CAG (Gln) codon in position 1 comprises an open reading frame of 1230 bp and encodes a 410 amino acid enzyme.
- pAB489-PhyM9 is the gene encoding the E. coli phytase variant PhyM9 with the A. niger phytase signal sequence (MGVSAVLLPLYLLSGVTS) Mullaney et al., Appl Microbiol Biotechnol, 1991, 35 (5), 611-614 Accession No. M94550) directly under the control of the cbh1 promoter.
- the 18 amino acid signal peptide from A. niger phytase was used to transform the phytase mutant of E. coli from Trichoderma reesei .
- pAB489-PhyM9 and pUC-PhyM9 were analogous to plasmid pAB489-PhyM3 and pUC-PhyM3 in Example 4 and thus contains, with the exception of the A. niger signal sequence and the specific E. coli phytase sequence the same genetic elements.
- the plasmid pUC-PhyM9 has been deposited under the accession number DSM 18718 on 18.10.2006.
- the plasmid pAB600-PhyM10 contains the E. coli phytase sequence with the changes of amino acids K74D, N139R, D142E and V200P. From the plasmids pAB490-PhyM2, pAB490-PhyM7 and pAB489-PhyM3 the gene coding for the phytase variant PhyM10 by the PCR method analogous to the principle of Tomic et al. (1990, Nucleic Acids Research, 18 (6), 1656 ) and Vallette et al. (1989, Nucleic Acids Research, 17 (2), 723-733 ) produced. The PCR product was cut with SpeI and PacI and inserted into the Spel and PacI cleavage sites in plasmid pAB600.
- the resulting plasmid pAB600-PhyM10 ( Fig. 6 ) was confirmed by mapping and sequencing.
- the E. coli phytase sequence is fused at the 3 'end of the CBHII gene fragment via a KexII site.
- the Kex sequence contains the following amino acids RTLVKR.
- the acetamidase ( amdS ) gene was isolated by the PCR method as Ascl Noul fragment and inserted into the Ascl / Stul cut plasmid pAB1280.
- the following primers were derived from the sequence information of the A. nidulans acetamidase gene: AmdS-AscI aggcgcgccctagtcatcattggataggcagattactcag (SEQ ID NO: 7) AmdS-NruI ggaattctcgcgaaaggcaacaaccagctcacccctgag (SEQ ID NO: 8)
- pAB600 The resulting plasmid has been designated pAB600 has been confirmed by mapping and sequencing.
- the phytase variant phyM10 gene was cloned into plasmid pAB2004.
- the plasmid pUC-PhyM10 is in Fig. 5 and has been deposited under the accession number DSM 18719 on 18.10.2006.
- the plasmid pPhy2005 contains the fusion from the A. niger var. Awamori acid phosphatase (ap) gene ( Piddington et al., 1993, Gene 133 (1), 55-62 ; Accession No. L02420) and the E. coli phytase gene gene using the codon usage of T. reesei (http://www.kazusa.or.jp/codon) (Dassa et al., 1990, Accession No. M58740). This results in an open reading frame encoding a 457 amino acid phytase variant with the first four (4) amino acids of the E.
- ap Awamori acid phosphatase
- the fusion of the A. niger var. Awamori acid phosphatase sequence and the E. coli phytase sequence generated using codon usage of T. reesei was synthesized and cloned into the plasmid pUC18.
- the resulting plasmid pPhy2005 was confirmed by sequencing.
- the plasmid is in Fig. 7 and has been deposited under the accession number DSM 18720 on 18.10.2006
- the sequence of the fusion phosphatase phytase from the plasmid pPhy2005 was cut with AvrII and Pacl and inserted into the Spel and Pacl cleavage sites after the T. reesei cbhI promoter in the plasmid pAB489.
- the resulting plasmid is called pAB-Phy2005 ( Fig. 8 ) and was mapped by restriction endonucleases and the phytase sequence confirmed by sequencing.
- Acid phosphatase gene fragment The acid phosphatase gene fragment with its signal sequence is directly under the control of the cbhl promoter.
- the 16 base pairs (CCGCGGACTGCGCATC atg) upstream of the start codon leading to the T. reesei promoter ( Shoemaker et al. 1983, Bio / Technology 1, 691-696 ) were changed to CCGCGGACTAGGCATC atg after incorporation of the AvrII-PacI fusion phosphatase phytase fragment.
- E. coli phytase gene The synthetic E. coli phytase gene is fused directly to the 3 'end of the acid phosphatase sequence into an open reading frame.
- cbhI terminator The 0.75 kb BamHI / StuI fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
- amdS gene The gene including its promoter and its terminator was isolated from Aspergillus nidulans VH1-TRSX6 and codes for acetamidase ( Hynes et al., 1983, Mol. Cell. Biol. 3: 1430-1439 ; Kelly and Hynes, 1985, EMBO J. 4: 475-479 ). Acetamidase allows the strain to grow using acetamide as the sole nitrogen source and this characteristic was used to select the transformants.
- cbhl 3 'fragment The fragment (1.7 kb, BamHI / EcoRI, starting 1.4 kb after the stop codon of the gene) was isolated from T. reesei ALKO2466.
- the strain ALKO2466 is derived from strain ALKO233 ( Harkki et al., 1991, Enzymes Microb. Technol. 13: 227-233 ).
- the 3 'fragment, along with the promoter region, is used to target the phytase expression cassette into the cbhl locus by homologous recombination.
- the 87 bp ap gene fragment coding for the 29 amino acids belonging to the C-terminus of the ap gene of the A. niger var. Awamori acid phosphatase was directly linked to that for the last amino acid (leucine).
- the fusion Phy2006 from the A. niger var. Awamori acid phosphatase sequence and the E. coli phytase sequence generated using the codon usage of T. reesei was synthesized and cloned into the plasmid pUC18.
- the new sequence contained in the obtained plasmid pPhy2006 was confirmed by sequencing.
- the plasmid is in Fig. 9 and has been deposited under the accession number DSM 18721 on 18.10.2006.
- the construction and the cloning of the plasmid pAB-Phy2006 is identical to the preparation of the plasmid pAB-Phy2005 described in Example 7.
- T. reesei RH 3780d was separately transformed with the linearized expression cassettes isolated from the plasmids pAB489-PhyM3, pAB490-PhyM2, pAB490-PhyM7, pAB489-PhyM9, pAB600-PhyM10, pAB-Phy2005 and pAB-Phy2006.
- the techniques used to transform and manipulate T. reesei were as follows Penttilä et al. (1987, Gene 61: 155-164 ). The transformants were selected and purified twice by single spore isolation. Of all the transformants, those with the highest secretion efficiency were selected and reused in Example 10 to prepare enzyme material. The transformants used are listed in Table 2 below.
- genotype Transformant Wt (pKDa4) RH 31071 pKDa2 (V200Y; M1) RH 31068 PhyM2 RH 31575 PhyM3 RH 31545 PhyM7 RH 31507 PhyM9 RH 31686 PhyM10 RH 31898 Phy2005 RH 31676 Phy2006 RH 31677
- Transformants carrying the expression cassettes of Examples 2 to 8, and the transformants with the plasmid pKDa2 (M1) and pKDa4 (wild-type amino acid sequence E. coli -Phytase Dassa) from DE 10 2004 050 410 were grown in shake flasks with pH control on a DASGIP system on cellulase-inducing medium of the following composition: lactose 10.5% (w / v), DSG 5.25% (w / v), (NH 4 ) 2 SO 4 0.63% (w / v), tap water remainder, adjust pre-sterilization pH to 4.5. After inoculation, a pH ramp was brought to pH 3.3 in the shake flask over 5 h. The culture filtrates obtained after 6 days of controlled pH control at pH 3.3 were used for the determination of phytase activity and for the analysis of thermostability by differential scanning calorimetry (DSC) (Example 11).
- DSC differential scanning calori
- Example 10 A The transformants of Example 10 A) were grown in the medium of Example 10 A) in 30-1 bioreactors at a pH of 3.2 ⁇ 0.2, 200 to 400 rpm and 0.5 vvm aeration rate. At the end of the 6-day fermentation, the biomass was separated by filtration and the clear culture supernatant was concentrated by a factor of 6 by means of ultrafiltration (30 kDa cut-off membrane). The concentrate was sterile filtered and then used for the subsequent production of enzyme granules.
- the granules were prepared from the UF concentrates by drying in a spray granulator of the type ProCell5, Glatt Systemtechnik GmbH, Dresden, Germany.
- the pH of the UF concentrates was adjusted to pH 5.2 and prior to drying 50% (w / w) skimmed milk powder with 30% lactose content and 9.2% (w / w) Ca propionate, both additions based on the Total protein content of the UF concentrate, added.
- the mixture was dried through a 1.2 mm ⁇ two-component nozzle with a rising from 1.2 to 2.2 bar air pressure at 80 ° -90 ° C inlet air temperature.
- the activity yields in the drying were in the range of 89 to 99%.
- the granules thus prepared were used for the experiment in Example 12.
- Example 10A The samples of Example 10A were buffered by means of a PD-10 column (Pharmacia) in 100 mM sodium acetate buffer, pH 5.2. For this purpose, 1.5 ml of sample were applied and eluted with 3.5 ml of buffer according to the manufacturer's instructions. All DSC experiments were performed on a VP-DSC instrument (MicroCal Inc., Northampton, MA, USA) and the data was transferred to a PC using the Origin v7.0383 software (OriginLab Corp. Northampton, MA). USA).
- the samples were degassed for 20 minutes in a ThermoVac2 (MicroCal Inc., Northampton, MA; USA) and tempered to 25 ° C in a water bath prior to measurement.
- ThermoVac2 MicroCal Inc., Northampton, MA; USA
- Table 3 below shows the melting temperature Tm (in degrees Celsius) and the shift in the melting temperature ⁇ T (in degrees Celsius) of the mutant E. coli phytases prepared according to the above examples compared to the wild-type E. Coli phytase (Dassa et al., 1990) prepared with the same host system as the mutant E. coli phytase variants and an E. coli phytase with a non-inventive mutation (M1), also prepared with the same host system as the mutant E. coli according to the invention phytase variants.
- Enzyme granulates prepared according to Example 10B were premixed with wheat flour, type 550, to obtain 300 g of an enzyme-containing premix.
- This premix was at the Biotechnology Institute of the Research Institute for Food and Molecular Biotechnology, Kolding, Denmark, mixed with 15 kg of animal feed to ensure optimum dilution for incorporation into 285 kg of animal feed and a well-determined enzyme activity in the pelleted material.
- the amount of enzyme granules used resulted in a phytase activity of about 3 to 6 units of enzyme per gram of animal feed in the mixture before the test process.
- the 300 kg animal feeds treated in the pilot pelletizer have the composition of Table 4.
- Table 4 Composition of the animal feed to be pelleted component [%] wheat Ad 75 Hipro Soy 48 20 soybean oil 4.75 Vitamin / Minerals, Beta Avitren 90 0.25 enzyme premix * 300 g Contains phytase with genotype wt, PhyM1, PhyM2, PhyM3, PhyM7, PhyM9, Phy2005, or Phy2006
- Horizontal mixer with 700 l volume, 48 upm.
- the amount that is mixed is in the range 80 to 300 kg per h.
- a horizontal screw conveyor for emptying the mixer, whose conveying speed is adapted to the subsequent step.
- the conditioned material is pelleted by a Simon Heesen press and then air cooled in a perforated box through which the flow passes at 1500 m 3 h -1 .
- the pelleting press runs at 500 rpm at a power consumption of 7.5 kW, producing pellets of 3 x 20 mm.
- thermostability found in liquid measurements by DSC was also found in the "dry" pelleting experiments.
- the improvement in thermostability compared to the wild-type enzyme is not constant for all mutants over the entire measured temperature range.
- M2 showed the highest temperature stability in the DSC experiment.
- the best stabilization was achieved by mutation in an isolated region through the double mutation M2, which inserts an ionic bridge into helix D of E. coli phytase.
- the noninventive mutant M1 shows, as in the DSC experiment, a deterioration of the thermostability in comparison to the wild-type enzyme expressed in Trichoderma reesei .
- the Mutation M3 which in itself is not according to the invention, shows similar thermostability properties as the wild-type enzyme.
- the variant Phy2005 which carries only the N-terminal extension by the corresponding part of the acid phosphatase from A. niger var. Awamori , is not as stable at higher temperatures as Phy2006, which contains both N- and C-terminal extension , This suggests that these extensions may well contribute to the aggregation of E. coli phytase molecules analogous to dimerization of the acid phosphatase under normal conditions and thus improve thermostability.
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Abstract
Description
Die Erfindung betrifft ein rekombinantes DNA-Molekül, das ein Polypeptid mit Phytaseaktivität und erhöhter Temperaturstabilität und erhöhter proteolytischer Stabilität der Enzymaktivität codiert sowie das codierte Polypeptid als solches. Insbesondere betrifft die Erfindung ein rekombinantes DNA-Molekül, das ein Polypeptid mit Phytaseaktivität und erhöhter Temperaturstabilität und erhöhter proteolytischer Stabilität der Enzymaktivität codiert, wobei die DNA-Sequenz durch eine Variation der reifen Wildtyp-E. coli-Phytase erhalten worden ist, wobei definierte Aminosäurepositionen im Vergleich zur Wildtypsequenz modifiziert sind bzw. N- und/oder C-terminale Extensionen vorhanden sind. Die Erfindung betrifft ferner ein Verfahren zur Expression der rekombinanten Phytase sowie deren Verwendung in der Lebensmittel- und Futtermitteltechnologie.The invention relates to a recombinant DNA molecule which encodes a polypeptide with phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity and the encoded polypeptide as such. More particularly, the invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity, wherein the DNA sequence has been obtained by a variation of wild-type mature E. coli phytase having defined amino acid positions are modified in comparison to the wild-type sequence or N- and / or C-terminal extensions are present. The invention further relates to a method for the expression of recombinant phytase and their use in food and feed technology.
Phytinsäure oder Myoinositol-1,2,3,4,5,6-hexakisdihydrogenphosphat (abgekürzt Myoinositol-Hexakisphosphat) ist die Hauptquelle von Inositol und die primäre Speicherform von Phosphat in Pflanzensamen. In den Samen von Hülsenfrüchten liegt etwa 70% des Phosphatgehalts als ein gemischtes Kalium-, Magnesium- und Calciumsalz der Phytinsäure vor. Samen, Getreidekörner und Hülsenfrüchte sind wichtige Komponenten von Lebens- und Futtermittelzubereitungen, insbesondere von Tierfutterpräparaten; aber auch in der menschlichen Nahrung gewinnen Getreide- und Hülsenfrüchte zunehmend an Bedeutung. Die Phosphateinheiten der Phytinsäure binden als Komplex zweiwertige und dreiwertige Kationen wie Metallionen, d.h. ernährungsphysiologisch wichtige Ionen wie Calcium, Eisen, Zink und Magnesium sowie die Spurenelemente Mangan, Kupfer und Molybdän. Daneben bindet Phytinsäure auch zu einem gewissen Ausmaß Proteine durch elektrostatische Wechselwirkung.Phytic acid or
Phytinsäure und ihre Salze, die Phytate, werden oft nicht metabolisiert, da sie aus dem Magen-Darm-Trakt nicht absorbiert werden, d.h. weder der darin enthaltene Phosphor noch die chelatierten Metallionen, noch die gebundenen Proteine sind ernährungsphysiologisch verfügbar.Phytic acid and its salts, the phytates, are often not metabolized because they are not absorbed from the gastrointestinal tract, i. neither the phosphorus contained therein nor the chelated metal ions nor the bound proteins are nutritionally available.
Da Phosphor ein wesentliches Element für das Wachstum aller Organismen ist, müssen Lebensmittel und Futtermittel mit anorganischem Phosphat supplementiert werden. Sehr oft müssen auch die ernährungsphysiologisch essenziellen Ionen wie Eisen und Calcium supplementiert werden. Ferner wird der ernährungsphysiologische Wert jeder Diät vermindert, da Proteine durch Phytinsäure gebunden werden. Folglich wird Phytinsäure oft als Anti-Nährwertfaktor bezeichnet.Since phosphorus is an essential element for the growth of all organisms, food and feed must be supplemented with inorganic phosphate. Very often, the nutritionally essential ions such as iron and calcium must be supplemented. Furthermore, the nutritional value of each diet is reduced because proteins are bound by phytic acid. Consequently, phytic acid is often referred to as an anti-nutritive factor.
Weiterhin wird aufgrund fehlender Metabolisierung von Phytinsäure der Phosphor des Phytats über den Magen-Darm-Trakt der Tiere ausgeschieden, was zu einer unerwünschten Phosphatverunreinigung der Umwelt führt, in deren Folge es beispielsweise zur Eutrophierung von Gewässern und zu exzessivem Algenwachstum kommen kann.Furthermore, due to lack of metabolism of phytic acid, the phosphorus of the phytate is excreted through the gastrointestinal tract of the animals, resulting in undesirable phosphate contamination of the environment, as a result of which, for example, eutrophication of waters and excessive algal growth may occur.
Phytinsäure oder Phytate (diese Ausdrücke werden im Folgenden synonym verwendet außer anders angegeben) können durch Phytasen abgebaut werden. Phytasen katalysieren die Hydrolyse von Phytat in Myo-Inositol und/oder Mono-, Di-, Tri-, Tetra- und/oder Pentaphosphate sowie anorganisches Phosphat. Man unterscheidet zwei verschiedene Arten von mikrobiellen Phytasen: 1) 3-Phytase/Myo-Inositolhexaphosphat-3-phosphohydrolase, EC 3.1.3.8; 2) 6-Phytase/Myo-Inositolhexaphosphat-6-phosphohydrolase, EC 3.1.3.26. Die 3-Phytase hydrolisiert bevorzugt zuerst die Esterbindung in der 3-Position, die 6-Phytase bevorzugt zuerst die Esterbindung in der 6-Position. Phytinsäure enthaltende Pflanzensamen enthalten endogene Phytaseenzyme. Bei deren Aufnahme sind die Phytate in Lebensmitteln bzw. Futtermitteln theoretisch durch die endogenen Pflanzen-Phytasen, durch Phytasen aus der Darmflora und durch Phytasen aus der Darmschleimhaut hydrolisierbar. In der Praxis ist jedoch das Hydrolysepotenzial der endogenen Pflanzen-Phytasen und der im Darm vorkommenden Phytasen, sofern vorhanden, bei weitem nicht ausreichend, um signifikant die Bioverfügbarkeit des in den Phytaten gebundenen Phosphors zu gewährleisten. Daher werden Lebens- bzw. Futtermitteln häufig exogene Phytasen zugesetzt.Phytic acid or phytates (these terms are used synonymously below unless otherwise specified) can be degraded by phytases. Phytases catalyze the hydrolysis of phytate into myo-inositol and / or mono-, di-, tri-, tetra- and / or pentaphosphates as well as inorganic phosphate. There are two different types of microbial phytases: 1) 3-phytase / myo-inositol hexaphosphate-3-phosphohydrolase, EC 3.1.3.8; 2) 6-phytase / myo-inositol hexaphosphate-6-phosphohydrolase, EC 3.1.3.26. The 3-phytase preferably hydrolyzes first the ester bond in the 3-position, the 6-phytase prefers first the ester bond in the 6-position. Phytic acid-containing plant seeds contain endogenous phytase enzymes. Upon ingestion, the phytates in food or feed are theoretically hydrolyzed by the endogenous plant phytases, by phytases from the intestinal flora and by phytases from the intestinal mucosa. In practice, however, the hydrolysis potential of the endogenous plant phytases and intestinal phytases, if present, is far from sufficient to significantly ensure the bioavailability of the phosphorus bound in the phytates. Therefore, food and feed are often added to exogenous phytases.
Phytasen können durch Pflanzen sowie durch Mikroorganismen produziert werden. Unter den Mikroorganismen sind Phytase produzierende Bakterien sowie Phytase produzierende Pilze und Hefen bekannt.Phytases can be produced by plants as well as by microorganisms. Among the microorganisms, phytase-producing bacteria and phytase-producing fungi and yeasts are known.
Die natürlich vorkommenden Phytase-Produzenten haben jedoch den Nachteil, dass Phytase nur in bestimmten Mengen und mit definierten Eigenschaften gebildet wird. Wie vorstehend dargelegt besteht jedoch ein erhöhter Bedarf an Phytase insbesondere für die Lebensmittel- und Futtermittelindustrie.However, the naturally occurring phytase producers have the disadvantage that phytase is formed only in certain quantities and with defined properties. However, as stated above, there is an increased need for phytase especially for the food and feed industry.
Obgleich in der Lebensmittel- und Futtermittelindustrie ein erhöhter Bedarf an Phytase besteht und die Verwendung von Phytase vorteilhaft sein kann, haben nur wenige der bekannten Phytasen eine breite Akzeptanz in der Lebensmittel- und Futtermittelindustrie gefunden. Typische Bedenken betreffen die relativ hohen Herstellungskosten und/oder schlechte Stabilität bzw. Aktivität des Enzyms in der gewünschten Anwendungsumgebung. Weiterhin muss ein derartiges Enzym eine Reihe von Kriterien erfüllen, um industriell einsetzbar zu sein. Diese umfassen eine hohe spezifische Gesamtaktivität, ein niedriges pH-Optimum, Resistenz gegenüber gastrointestinalen Proteasen sowie Temperaturstabilität bzw. Thermostabilität. Die Temperaturstabilität ist eine wichtige Voraussetzung für eine erfolgreiche industrielle Anwendung, da beispielsweise in Pelletierprozessen Enzyme Temperaturen zwischen 60°C und 95°C ausgesetzt werden. Alle bekannten mikrobiellen Phytasen entfalten sich bei Temperaturen zwischen 56°C und 78°C (Lehman et al., 2000), wodurch sie ihre Aktivität verlieren. Daher besteht insbesondere ein Bedarf an Phytasen, die auch bei erhöhten Temperaturen eine technologisch ausreichende Aktivität besitzen, bzw. nicht inaktiviert werden.Although there is an increased demand for phytase in the food and feed industry and the use of phytase may be beneficial, only a few of the known phytases have found wide acceptance in the food and feed industry. Typical concerns concern the relatively high production cost and / or poor stability or activity of the enzyme in the desired environment of use. Furthermore, such an enzyme must meet a number of criteria in order to be industrially useful. These include high specific total activity, a low pH optimum, resistance to gastrointestinal proteases and temperature stability or thermal stability. Temperature stability is an important prerequisite for successful industrial application, since enzymes are exposed to temperatures between 60 ° C and 95 ° C in pelleting processes, for example. All known microbial phytases unfold at temperatures between 56 ° C and 78 ° C (Lehman et al., 2000), thereby losing their activity. Therefore, in particular, there is a need for phytases which, even at elevated temperatures, have a technologically sufficient activity or are not inactivated.
Der vorliegenden Erfindung liegt somit die Aufgabe zu Grunde, ein Polypeptid mit Phytaseaktivität bereit zu stellen, das eine erhöhte Thermostabilität aufweist bzw. bei erhöhten Temperaturen eine technologisch ausreichende Aktivität besitzt. Ferner soll das Polypeptid mit Phytaseaktivität auch eine erhöhte proteolytische Stabilität besitzen.The present invention is therefore based on the object to provide a polypeptide with phytase activity, which has an increased thermal stability or at elevated temperatures has a technologically sufficient activity. Furthermore, the polypeptide with phytase activity should also have increased proteolytic stability.
Das Polypeptid soll ökonomisch produziert werden können. Insbesondere soll die Phytase thermostabiler sein als das Wild-Typ-Enzym. Die Phytase soll ferner die wesentlichen Eigenschaften der natürlichen E. coli-Wildtyp-Phytase beibehalten, sich jedoch durch eine verbesserte Thermostabilität auszeichnen. Zu den wesentlichen Eigenschaften der natürlichen Wildtyp-Phytase zählen insbesondere die Fähigkeit, die Verfügbarkeit von Phosphat in-vivo und in-vitro zu verbessern durch seine Aktivität als Phytase wie auch als Phosphatase, sein pH-Optimum im sauren mit hoher Restaktivität im stark sauren, sowie die Eignung als Backhilfsmittel.The polypeptide should be able to be produced economically. In particular, the phytase should be more thermostable than the wild-type enzyme. The phytase should also retain the essential properties of the natural E. coli wild-type phytase, but be characterized by improved thermal stability. The essential properties of the wild type natural phytase include in particular the ability to improve the availability of phosphate in vivo and in vitro by its activity as a phytase as well as a phosphatase, its pH optimum in the acidic with high residual activity in strongly acidic, as well as the suitability as baking aid.
Der vorliegenden Erfindung liegt ferner die Aufgabe zu Grunde, ein Gen für ein Polypeptid mit Phytaseaktivität mit erhöhter Thermostabilität sowie erhöhter proteolytischen Stabilität bereit zu stellen. Das Polypeptid soll ökonomisch und kostengünstig zu produzieren sein. Insbesondere soll die Expression des Polypeptids in eukaryotischen Mikroorganismen zu einem Polypeptid mit erhöhter Thermostabilität verglichen mit der ähnlich hergestellten Wildtyp-Phytase führen. Bereitgestellt werden sollen ferner die das Polypeptid codierenden DNA-Sequenzen, entsprechende DNA-Konstrukte und Vektoren sowie eine Quelle für das rekombinante Enzym, die für die kommerzielle Verwendung für Nahrungs- und Futtermittel und in industriellen Prozessen geeignet ist und das erfindungsgemäße Enzym enthaltende Zusammensetzungen.It is a further object of the present invention to provide a gene for a polypeptide having phytase activity with increased thermostability and increased proteolytic stability. The polypeptide should be economical and inexpensive to produce. In particular, expression of the polypeptide in eukaryotic microorganisms should result in a polypeptide with increased thermostability compared to the similarly prepared wild-type phytase. Also provided are the DNA sequences encoding the polypeptide, corresponding DNA constructs and vectors, and a source of recombinant enzyme suitable for commercial use in food processing. and feed and in industrial processes, and the enzyme-containing compositions of the invention.
Es wurde nun überraschenderweise gefunden, dass Mutationen an bestimmten Positionen der E. coli-Wildtyp-Phytasesequenz zu einer intrinsischen Erhöhung der Thermostabilität bzw. Temperaturstabilität sowie zu einer Erhöhung der proteolytischen Stabilität des Proteins Phytase führen, ohne die sonstigen Wirkungen und wesentlichen Eigenschaften der Wildtyp-E. coli-Phytase nachteilig zu beeinflussen.It has now surprisingly been found that mutations at certain positions of the E. coli wild-type phytase sequence lead to an intrinsic increase in the thermostability or temperature stability and to an increase in the proteolytic stability of the phytase protein, without the other effects and essential properties of the wild type. To adversely affect E. coli phytase.
Überraschenderweise wurde gefunden, dass eine Mutation bei Position 74 (K74D) der Aminosäurensequenz, und/oder eine Kombination von Mutationen bei den Positionen 139 (N139R) und 142 (D142E), und/oder eine Kombination von Mutationen bei den Positionen 145 (L1451) und 198 (L1981) und/oder eine Mutation bei Position 200 (V200P) der Wildtyp-Phytase von E. coli sowie Kombinationen dieser Mutationen zu einer verbesserten Thermostabilität des Proteins Phytase führen ohne die günstigen Wirkungen und wesentlichen Eigenschaften der Wildtyp-E. coli-Phytase zu beeinträchtigen. Weiterhin wurde gefunden, dass die Ergänzung der E. coli-Phytase durch Sequenzen der sauren Phosphatase aus Aspergillus niger var. awamori am N-terminalen oder C-terminalen Ende, bzw. an den N- und C-terminalen Enden, auch in Verbindung mit den vorhergenannten Mutationen, zu einer Verbesserung der Thermostabilität und der proteolytischen Stabilität des Enzyms führen.Surprisingly, it has been found that a mutation at position 74 (K74D) of the amino acid sequence, and / or a combination of mutations at positions 139 (N139R) and 142 (D142E), and / or a combination of mutations at positions 145 (L1451) and 198 (L1981) and / or a mutation at position 200 (V200P) of the wild-type phytase of E. coli as well as combinations of these mutations lead to an improved thermostability of the protein phytase without impairing the beneficial effects and essential properties of the wild-type E. coli phytase. Furthermore, it has been found that the addition of E. coli -Phytase by sequences of acid phosphatase from Aspergillus niger var. Awamori at the N-terminal or C-terminal end, or at the N- and C-terminal ends, in connection with the aforementioned mutations, lead to an improvement in the thermal stability and the proteolytic stability of the enzyme.
Die Erfindung betrifft somit ein rekombinantes DNA-Molekül das nach Expression in einer prokaryotischen oder eukaryotischen Wirtszelle ein Polypeptid mit Phytaseaktivität codiert, wobei das rekombinante DNA-Molekül eine DNA-Sequenz, ausgewählt aus
- a) DNA-Sequenzen, die ein Polypeptid mit Phytaseaktivität codieren, das durch Variation der reifen Wildtyp-E. coli-Phytasesequenz erhalten worden ist, wobei die Variation ausgewählt ist aus
- i) der Mutation Lysin → Asparaginsäure an Position 74 (K74D), und/oder
- ii) einer Kombination der Mutationen Asparagin → Arginin an Position 139 (N139R) und Asparaginsäure → Glutaminsäure an Position 142 (D142E), und/oder
- iii) einer Kombination der Mutationen Leucin → Isoleucin an Position 145 (L1451) und Leucin → Isoleucin an Position 198 (L1981), und/oder
- iv) einer Mutation Valin → Prolin an Position 200 (V200P), und/oder
- v) einer N-terminalen oder C-terminalen oder einer N- und C-terminalen Anfügung eines Sequenzabschnitts der sauren Phosphatase von Aspergillus niger var. awamori, oder der Phytase von Aspergillus niger,
- b) DNA-Sequenzen, die eine Homologie von 70% bis 100% zu den Sequenzen gemäß a) aufweisen,
- c) DNA-Sequenzen, die aufgrund der Degeneriertheit des genetischen Codes mit den Sequenzen gemäß a) und b) verwandt sind,
- a) DNA sequences encoding a polypeptide with phytase activity obtained by variation of the wild-type mature E. coli phytase sequence, the variation being selected from
- i) the mutation lysine → aspartic acid at position 74 (K74D), and / or
- ii) a combination of the mutations asparagine → arginine at position 139 (N139R) and aspartic acid → glutamic acid at position 142 (D142E), and / or
- iii) a combination of the mutations leucine → isoleucine at position 145 (L1451) and leucine → isoleucine at position 198 (L1981), and / or
- iv) a mutation valine → proline at position 200 (V200P), and / or
- v) an N-terminal or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. awamori, or the phytase of Aspergillus niger,
- b) DNA sequences having a homology of 70% to 100% to the sequences according to a),
- c) DNA sequences related to the sequences according to a) and b) due to the degeneracy of the genetic code,
Mehrere Phytasen aus E. coli sind in der Literatur beschrieben, z. B. das für eine Phytase codierende appA-Gen aus E. coli K-12 (
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Es ist praktisch nicht möglich, Vorhersagen über die Wirkung einer bzw. mehrerer Mutationen auf das Verhalten der Enzymaktivität unter Bedingungen erhöhter Temperatur zu treffen. Generell erfolgt bei höheren Temperaturen eine Denaturierung bzw. Entfaltung der Sekundär- und Tertiärstruktur des Proteins.It is virtually impossible to make predictions about the effect of one or more mutations on the behavior of the enzyme activity under conditions of elevated temperature. Generally, at higher temperatures, denaturation or unfolding of the secondary and tertiary structure of the protein occurs.
Die Temperaturstabilität bzw. Thermostabilität von Proteinen ist abhängig von einer Vielzahl an Wechselwirkungen. Die Konformation von Proteinen wird durch eine große Anzahl schwacher Wechselwirkungen aufrechterhalten. Die Stabilisierung kann alle Hierarchieebenen der Proteinstruktur umfassen: Lokale Packung der Polypeptidekette, Sekundär- und Supersekundärstrukturelemente, Domänen und Untereinheiten eines multimeren Proteins. Es gibt zahlreiche Gründe, die hinsichtlich der erhöhten Temperaturstabilität eines Proteins berichtet werden, wobei die häufigsten (a) die Zusammenlagerung von Ionenpaaren innerhalb und/oder zwischen Untereinheiten; (b) verbesserte Packung des hydrophoben Kerns (van-der-Waals-Wechselwirkungen); (c) zusätzliche Netzwerke aus Wasserstoffbrücken; (d) verstärkte Neigung zur Sekundärstrukturbildung; (e) erhöhte Dipolstabilisierung innerhalb der Helix; (f) eine vergrößerte polare Oberfläche; (g) eine verringerte Anzahl und ein verringertes Gesamtvolumen von Kavitäten; (h) die Verringerung von konformationeller Spannung (Schleifenstabilisierung) und (i) Widerstandsfähigkeit gegenüber chemischer Modifikation (Oxidation von Methioninresten und/oder Desaminierung von Asparagin- und Glutaminresten), sind.The temperature stability or thermostability of proteins depends on a variety of interactions. The conformation of proteins is maintained by a large number of weak interactions. Stabilization may include all levels of protein structure hierarchy: local packaging of the polypeptide chain, secondary and supersecond structural elements, domains and subunits of a multimeric protein. There are many reasons reported for increased temperature stability of a protein, with the most common being (a) the assembly of ion pairs within and / or between subunits; (b) improved packing of the hydrophobic core (van der Waals interactions); (c) additional hydrogen-bonded networks; (d) increased tendency to secondary structure formation; (e) increased dipole stabilization within the helix; (f) an enlarged polar surface; (g) a reduced number and a reduced total volume of cavities; (h) reducing conformational stress (loop stabilization) and (i) resistance to chemical modification (oxidation of methionine residues and / or deamination of asparagine and glutamine residues).
Im Stand der Technik sind keine Hinweise dahingehend bekannt, wie und in welcher Art die E. coli-Wildtyp-Phytasesequenz gezielt zu verändern ist, um eine Erhöhung der Thermostabilität zu erzielen. Der Stand der Technik enthält ferner keine Hinweise auf die vorstehend genannten Mutationen in der E. coli-Phytasesequenz.In the prior art, no information is known as to how and in what manner the E. coli wild-type phytase sequence is to be changed specifically in order to achieve an increase in the thermal stability. The prior art also contains no indications of the aforementioned mutations in the E. coli phytase sequence.
Der Stand der Technik enthält insbesondere keine Hinweise dahingehend, dass eine Mutation bei Position 74 (K74D) der Aminosäuresequenz oder eine Kombination von Mutationen bei den Positionen 139 (N139R) und 142 (D142E) oder eine Kombination von Mutationen bei den Positionen 145 (L1451) und 198 (L198I) oder eine Mutation bei Position 200 (V200P) der Wildtyp-Phytase von E. coli oder Kombinationen dieser Mutationen zu einer verbesserten Thermostabilität des Proteins Phytase führen ohne die günstigen Wirkungen und wesentlichen Eigenschaften der Wildtyp-E. coli-Phytase zu beeinträchtigen. Ferner enthält der Stand der Technik keine Hinweise darauf, dass die Ergänzung der E. coli-Phytasesequenz durch Sequenzen der sauren Phosphatase aus Aspergillus niger var. awamori am N-terminalen oder C-terminalen Ende, bzw. N- und C-terminalen Enden, auch in Verbindung mit den vorher genannten Mutationen, zu einer Verbesserung der Thermostabilität des Enzyms führt.In particular, the prior art contains no evidence that a mutation at position 74 (K74D) of the amino acid sequence or a combination of mutations at positions 139 (N139R) and 142 (D142E) or a combination of mutations at positions 145 (L1451) and 198 (L198I) or a mutation at position 200 (V200P) of the wild-type phytase of E. coli or combinations of these mutations lead to improved thermostability of the phytase protein without the beneficial effects and essential properties of the wild-type E. coli phytase. Furthermore, the prior art contains no evidence that the addition of E. coli -Phytasesequenz by sequences of acid phosphatase from Aspergillus niger var. awamori at the N-terminal or C-terminal end, or N- and C-terminal ends, also in conjunction with the aforementioned mutations, to an improvement in the thermostability of the enzyme leads.
Bevorzugte erfindungsgemäße rekombinante DNA-Moleküle mit den erfindungsgemäßen Variationen der reifen Wildtyp-E. coli-Phytasesequenz sind die Konstrukte PhyM2, PhyM3, PhyM7, PhyM9 und PhyM10. Sie sind in der nachstehenden Tabelle 1 dargestellt. Die Konstruktion PhyM1 wurde als Vergleichsprodukt aufgenommen.
Die folgenden Plasmide sind bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, am 18.10.2006 gemäß den Bedingungen des Budapester Vertrags hinterlegt worden.
Es war überraschend, dass Mutationen, die die hydrophobe Oberfläche des Proteins beeinflussen (PhyM3, PhyM7 und PhyM9), sowie Mutationen, die eine ionische Bindung in Helix D (PhyM2) einfügen, bzw. den Loop B4 über eine ionische Brücke stabilisieren (PhyM7) signifikant zur Erhöhung der Thermostabilität der E. coli-Phytase beitragen.It was surprising that mutations that influence the hydrophobic surface of the protein (PhyM3, PhyM7, and PhyM9), as well as mutations that introduce an ionic bond into helix D (PhyM2) or loop B4 stabilize via an ionic bridge (PhyM7) contribute significantly to increasing the thermostability of E. coli phytase.
Auch das Anfügen von Sequenzteilen der Aspergillus niger var. awamori sauren Phosphatase, die bei der sauren Phosphatase zu einer Di- und Tetramerbildung führen, hatte stabilisierende Effekte bei der E. coli-Phytase. Entsprechendes gilt für die Anfügung von Sequenzteilen der Phytase von Aspergillus niger. The addition of sequence parts of Aspergillus niger var. Awamori acid phosphatase, which lead to the formation of di- and tetramers in acid phosphatase, had stabilizing effects on E. coli phytase. The same applies to the addition of sequence parts of the phytase of Aspergillus niger.
Die erfindungsgemäßen Polypeptide mit Phytaseaktivität zeigen verglichen mit dem E. coli-Wildtyp-Enzym eine erhöhte Temperatur bzw. Thermostabilität ihrer Enzymaktivität. Diese Verbesserung der Thermostabilität kann in Flüssigkeit mittels Differential Scanning Calorimetry (DSC) gemessen werden. Insbesondere zeigt sich die Verbesserung der Thermostabilität an der stark erhöhten Restaktivität der Enzymmutanten gegenüber dem Wildtyp-Enzym, wenn sie der thermischen Belastung ausgesetzt werden, wie sie bei der Pelletierung von Tierfutter verwendet wird. Ferner besitzen die erfindungsgemäßen Polypeptide auch eine erhöhte Stabilität gegenüber proteolytischem Abbau wie er im speziellen im Magen von Geflügel und Monogastriern auftritt. Da die Phytase bei der Tierernährung speziell während der Magenpassage das Phosphat aus Phytinsäure und deren Abbauprodukten freisetzen muss, ist diese Stabilität von besonderem Interesse um die Dosage des Enzyms möglichst klein zu halten. Aufgrund der erhöhten Temperatur- und/oder proteolytischen Stabilität eignen sich die erfindungsgemäßen Phytasen insbesondere für Anwendungen, bei denen erhöhte Temperaturbedingungen auftreten bzw. bei denen mit einem erhöhten proteolytischen Abbau zu rechnen ist. Anwendungsbeispiele hierfür sind die Herstellung des Enzyms, bei der es vor Proteasen des Wirtsstammes geschützt werden muß, die Herstellung von pelletiertem Tierfutter, jedoch auch die Anwendung als Flüssigenzym bei der Post-pelleting Aufbringung auf das pelletierte Tierfutter. Eine erhöhte Temperaturstabilität erlaubt die frühere Aufbringung auf die noch nicht so weit abgekühlten Pellets, wodurch die Verdunstungszeit der aufgebrachten Flüssigkeit verkürzt wird. Auch mikrobiologisch bringt dies Vorteile, da dann der hohe Wassergehalt nur bei hohen Temperaturen kurzzeitig vorliegt, bei denen die meisten pathogenen Keime nicht wachsen können.The polypeptides according to the invention with phytase activity show an increased temperature or thermal stability of their enzyme activity compared with the E. coli wild-type enzyme. This improvement in thermal stability can be measured in liquid by differential scanning calorimetry (DSC). In particular, the improvement in thermostability is evidenced by the greatly increased residual activity of the enzyme mutants over the wild-type enzyme when exposed to the thermal stress used in the pelleting of animal feed. Furthermore, the polypeptides of the invention also have an increased stability to proteolytic degradation as it occurs especially in the stomach of poultry and monogastric. As the phytase in animal nutrition, especially during gastric passage, the phosphate from phytic acid and their degradation products must release, this stability is of particular interest to keep the dosage of the enzyme as small as possible. Due to the increased temperature and / or proteolytic stability, the phytases according to the invention are particularly suitable for applications in which elevated temperature conditions occur or in which increased proteolytic degradation is to be expected. Examples of use for this purpose are the preparation of the enzyme, in which it must be protected from proteases of the host strain, the production of pelleted animal feed, but also the use as a liquid enzyme in the post-pelleting application to the pelleted animal feed. An increased temperature stability allows the earlier application to the not so far cooled pellets, whereby the evaporation time of the applied liquid is shortened. This also has microbiological benefits, since then the high water content is present only briefly at high temperatures, at which most pathogenic germs can not grow.
Die erfindungsgemäße eine Phytase codierende DNA-Sequenz umfasst mindestens eine der folgenden Mutationen bzw. Kombinationen von Mutationen und/oder sowie N- oder C- bzw. N- und C-terminalen Anfügungen des codierten Polypeptids:
- i) die Mutation Lysin → Asparaginsäure an Position 74 (K74D), und/oder
- ii) eine Kombination der Mutationen Asparagin → Arginin an Position 139 (N139R) und Asparaginsäure → Glutaminsäure an Position 142 (D142E), und/oder
- iii) eine Kombination der Mutationen Leucin → Isoleucin an Position 145 (L1451) und Leucin → Isoleucin an Position 198 (L198I), und/oder
- iv) eine Mutation Valin → Prolin an Position 200 (V200P), und/oder
- v) eine N-terminale oder C-terminale oder eine N- und C-terminale Anfügung eines Sequenzabschnitts der sauren Phosphatase von Aspergillus niger var. awamori oder der Phytase von Aspergillus niger.
- i) the mutation lysine → aspartic acid at position 74 (K74D), and / or
- ii) a combination of the mutations asparagine → arginine at position 139 (N139R) and aspartic acid → glutamic acid at position 142 (D142E), and / or
- iii) a combination of the mutations leucine → isoleucine at position 145 (L1451) and leucine → isoleucine at position 198 (L198I), and / or
- iv) a mutation valine → proline at position 200 (V200P), and / or
- v) an N-terminal or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. awamori or the phytase of Aspergillus niger.
Die erfindungsgemäße Phytasesequenz enthält dabei bevorzugt mindestens eine und besonders bevorzugt mindestens zwei der vorstehend genannten Variationen. Beliebige Kombinationen der oben genannten Mutationsvarianten sind möglich um die Thermostabilität der Phytase an die vorliegenden Gegebenheiten der speziellen Anwendung anzupassen. Eine bevorzugte Kombinationsmöglichkeit stellt die Kombination von i), ii) und iv) dar, die im Weiteren als Genotyp PhyM10 bezeichnet wird.The phytase sequence according to the invention preferably contains at least one and particularly preferably at least two of the abovementioned variations. Any combination of the above-mentioned mutation variants are possible in order to adapt the thermostability of the phytase to the prevailing conditions of the particular application. A preferred combination option is the combination of i), ii) and iv), which is referred to below as the genotype PhyM10.
Ferner kann zusätzlich zu den oder anstelle der beschriebenen Mutationen bzw. Kombinationen von Mutationen eine N-terminale oder C-terminale oder eine N- und C-terminale Anfügung eines Sequenzabschnitts der sauren Phosphatase von Aspergillus niger var. awamori vorhanden sein. Auch das alleinige Vorhandensein dieser Sequenzabschnitte führt bereits zu einer Erhöhung der Thermostabilität. Bei dem N-terminalen Abschnitt der sauren Phosphatase aus A. niger var. awamori handelt es sich um die ersten 51 Aminosäuren des reifen Proteins (FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS). Die letzten 4 Aminosäuren dieses Polypeptids ersetzen dabei die ersten 4 Aminosäuren (QSEP) der reifen E. coli Phytase. Auch das Anfügen von Teilen dieses Abschnitts der sauren Phosphatase ist möglich. Alternativ kann auch der N-terminale Teil oder Teile davon, der ersten 40 Aminosäuren des reifen Proteins der Aspergillus niger Phytase verwendet werden (SCDTVDQGYQ CFSETSHLWG QYAPFFSLAN ESVISPEV-PA). Die Anfügung an die E. coli Phytase kann auch an anderen Stellen als an der beschriebenen Position 4 erfolgen, solange die Aktivität des Enzyms erhalten bleibt. Zur C-terminalen Verlängerung wurden die letzten 29 Aminosäuren (LSFWWNYNTT TELNYRSSPI ACQEGDAMD) der sauren Phosphatase aus A. niger var. awamori direkt an das C-terminale Ende der E. coli Phytase fusioniert. Diese Fusion, bzw. Fusionen, können auch mit weiteren Mutationen in der E. coli Phytase einhergehen und sind nicht beschränkt auf die in dieser Anmeldung aufgeführten weiteren Mutationen. Durch die Mutation Lys43Cys würde die Möglichkeit einer Disulphidbrückenbildung zwischen dem neuen C-terminalen Teil und dem E. coli Phytase Kern ermöglicht und damit eine verbesserte Dimerisierung. Weitere Mutationen, die van der Waals, hydrophobe oder ionische intermolekulare Brücken begünstigen oder stabilisieren und damit die Thermostabilität erhöhen oder noch weiter verbessern können, sind möglich. Beispiele für die N- bzw. C-terminalen Sequenzverlängerungen sind die nachstehend beschriebenen Ausführungsformen Phy2005 und Phy2006.Furthermore, in addition to or instead of the described mutations or combinations of mutations, an N-terminal or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. Awamori may be present. Even the sole presence of these sequence sections already leads to an increase in the thermal stability. The N-terminal portion of the A. niger var. Awamori acid phosphatase is the first 51 amino acids of the mature protein (FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS). The last 4 amino acids of this polypeptide replace the first 4 amino acids (QSEP) of mature E. coli phytase. The addition of parts of this section of the acid phosphatase is possible. Alternatively, the N-terminal part or parts thereof, the first 40 amino acids of the mature protein of Aspergillus niger phytase can be used (SCDTVDQGYQ CFSETSHLWG QYAPFFSLAN ESVISPEV-PA). The appendix to the E. coli phytase may also occur elsewhere than at position 4 described as long as the activity of the enzyme is maintained. The C-terminal extension, the last 29 amino acids (LSFWWNYNTT TELNYRSSPI ACQEGDAMD) of the acid phosphatase from A. niger var were. Awamori directly to the C-terminal end of the E. coli phytase fused. This fusion, or mergers, may also be associated with further mutations in the E. coli phytase and are not limited to the other mutations listed in this application. The mutation Lys43Cys would allow the possibility of disulphide bridge formation between the new C-terminal part and the E. coli phytase nucleus allows thus improved dimerization. Other mutations that favor or stabilize van der Waals, hydrophobic or ionic intermolecular bridges and thus increase or even improve the thermal stability are possible. Examples of the N- or C-terminal sequence extensions are the embodiments described below Phy2005 and Phy2006.
Die Thermostabilität der erfindungsgemäßen Phytase kann in Handelsprodukten noch weiter erhöht werden, z. B. durch Zugabe von Stoffen in das Ultrafiltrationskonzentrat vor der Trocknung oder Granulation. Beispielsweise kann eine stabile Formulierung des erfindungsgemäßen Phytaseenzyms dadurch hergestellt werden, dass ein Gemisch aus einer flüssigen Enzymlösung auf ein Füllmittel wie Maltodextrin gesprüht wird und dann das Gemisch getrocknet wird, oder der flüssigen Enzymlösung wird vor dem Trocknen 20% - 60% Magermilchpulver (w/w bezogen auf Gesamtprotein der Enzymlösung) und/oder 1 % - 5% Calciumpropionat (w/w bezogen auf Gesamtprotein der Enzymlösung) zugesetzt und der pH-Wert auf einen definierten Wert eingestellt, insbesondere auf pH 5,2 ± 0,5. Die Verringerung der Feuchtigkeit und die Bindungswechselwirkungen der Phytase mit dem Füllmittel schützen das Enzym zusätzlich zu seiner in seiner Struktur definierten Stabilität vor Umwelteinflüssen wie Temperaturextrema, die während der Herstellung des Futtermittels auftreten können. Trockene und flüssige Formulierungen können weiter stabilisiert werden, wenn die Aktivität potenzieller proteolytischer Enzyme verringert wird, die als Nebenprodukte in dem Flüssigfermentationsgemisch vorhanden sein können, das zur Herstellung des erfindungsgemäßen Enzyms verwendet wird.The thermostability of the phytase according to the invention can be further increased in commercial products, for. B. by adding substances into the ultrafiltration concentrate before drying or granulation. For example, a stable formulation of the phytase enzyme of the present invention can be prepared by spraying a mixture of a liquid enzyme solution onto a filler such as maltodextrin and then drying the mixture, or 20% -60% skimmed milk powder (w / o) before drying the liquid enzyme solution. w based on total protein of the enzyme solution) and / or 1% -5% calcium propionate (w / w based on total protein of the enzyme solution) was added and the pH was adjusted to a defined value, in particular to pH 5.2 ± 0.5. The reduction of moisture and the binding interactions of the phytase with the filler protect the enzyme, in addition to its stability defined in its structure, from environmental influences such as temperature extremes that may occur during the manufacture of the feed. Dry and liquid formulations can be further stabilized by reducing the activity of potential proteolytic enzymes which may be present as by-products in the liquid fermentation mixture used to prepare the enzyme of the present invention.
Die der erfindungsgemäß mutierten Phytasesequenz entsprechende DNA-Sequenz kann unter Verwendung beliebiger Codon usages realisiert werden. So kann beispielsweise die Codon usage des zur Expression verwendeten Mikroorganismus verwendet werden, es kann aber auch die E. coli-Codon-usage bzw. eine Variante davon verwendet werden. Ferner kann die erfindungsgemä-βe mutierte E. coli-Phytasesequenz weitere Sequenzvariationen enthalten. Dabei können beliebige Variationen zusätzlich zu den vorstehend beschriebenen Mutationen vorgenommen werden, solange die Eigenschaft der erhöhten Temperaturstabilität nicht nachteilig beeinträchtigt wird und solange die enzymatische Aktivität und weitere wesentliche Eigenschaften der E. coli-Wildtyp-Phytase beibehalten werden.The DNA sequence corresponding to the phytase sequence mutated according to the invention can be realized using any codon usages. For example, the codon usage of the microorganism used for expression can be used, but it can also be used the E. coli codon usage or a variant thereof. Furthermore, the mutated E. coli phytase sequence according to the invention may contain further sequence variations. Any variations may be made in addition to the mutations described above as long as the property of increased temperature stability is not adversely affected and as long as the enzymatic activity and other essential properties of the E. coli wild type phytase are maintained.
Entsprechende Variationen sind einem Fachmann auf dem Gebiet der Technik der rekombinanten DNA gut bekannt und umfassen die vorstehenden Mutationen sowie die nachstehend dargelegten beispielhaften Variationen.Corresponding variations are well known to a person skilled in the art of recombinant DNA technology and include the above mutations as well as the exemplary variations set forth below.
Erfindungsgemäß können Additions- und/oder Deletionsmoleküle des erfindungsgemäß modifizierten Polypeptids verwendet werden. So kann das erfindungsgemäß modifizierte Polypeptid mit Phytaseaktivität durch Anfügen weiterer Sequenzen am N-terminalen und/oder C-terminalen Ende verlängert werden. Dadurch können Hybridmoleküle hergestellt werden, die weitere vorteilhafte Eigenschaften besitzen. Beispielsweise können Fusionsproteine bzw. nativ in hohem Maße sekretierte Proteine angefügt werden, wodurch die Sekretionseffizienz verbessert wird. Bevorzugt wird dazu ein Teil des CBH2-Proteins aus Trichoderma reesei, von Aminosäure Met 1 bis Ser 86, verwendet.According to the invention, addition and / or deletion molecules of the polypeptide modified according to the invention can be used. Thus, the modified polypeptide with phytase activity according to the invention can be extended by adding further sequences at the N-terminal and / or C-terminal end. As a result, hybrid molecules can be produced which have further advantageous properties. For example, fusion proteins or native highly secreted proteins can be added, thereby improving secretion efficiency. A part of the CBH2 protein from Trichoderma reesei, from
Erfindungsgemäß können auch Sequenzabschnitte des Polypeptids mit Phytaseaktivität deletiert werden, solange die Eigenschaft der erhöhten Temperaturstabilität unter Beibehaltung der Phytaseaktivität nicht beeinträchtigt wird.Sequence sections of the polypeptide with phytase activity can also be deleted according to the invention as long as the property of increased temperature stability is not impaired while maintaining the phytase activity.
Die Mutationen, Elongationen und Verkürzungen können in an sich bekannter Weise nach auf dem Fachgebiet gut bekannten Verfahren durchgeführt werden.The mutations, elongations and truncations can be carried out in a manner known per se according to methods well known in the art.
Die vorstehend in Betracht gezogenen Veränderungen des Polypeptids mit Phytaseaktivität entsprechen entsprechenden Mutationen bzw. Modifikationen des dazugehörigen DNA-Moleküls. Erfindungsgemäß werden dabei auch solche Sequenzen in Betracht gezogen, die unter relaxierten oder stringenten Bedingungen mit den erfindungsgemäßen Sequenzen hybridisieren. Als stringente Bedingungen gelten: Hybridisierung bei 65°C, 18 h in Dextransulfat-Lösung (GenescreenPlus, DuPont), danach waschen der Filter jeweils 30 min zuerst mit 6 x SSC, zweimal 2 x SSC, zweimal 2 x SSC mit 0, 1 % SDS und anschließend mit 0,2 x SSC bei 65°C (Membrane transfer and detection methods, Amersham).The modifications of the polypeptide having phytase activity contemplated above correspond to corresponding mutations or modifications of the associated DNA molecule. According to the invention are also such Contemplated sequences that hybridize under relaxed or stringent conditions with the sequences of the invention. Stringent conditions are: hybridization at 65 ° C, 18 h in dextran sulfate solution (GenescreenPlus, DuPont), then wash the filter for 30 min each first with 6 x SSC, twice 2 x SSC, twice 2 x SSC with 0, 1% SDS followed by 0.2 x SSC at 65 ° C (membrane transfer and detection methods, Amersham).
Ferner betrifft die Erfindung auch solche Sequenzen, die mit der beanspruchten Nukleotidsequenz bzw. den beanspruchten Teilen davon eine Homologie von mindestens 70%, bevorzugter mindestens 80%, noch bevorzugter mindestens 90% und insbesondere mindestens 95% aufweisen, solange die jeweiligen Sequenzen zur Erhöhung der Temperaturstabilität des durch sie codierten Polypeptids mit Phytaseaktivität führen. Bevorzugt beträgt die Homologie 70 bis zu 100 Prozent. Die Homologie ist dabei als Grad der Identität definiert. Der Grad der Identität wird dabei bevorzugt so bestimmt, dass man die Anzahl der Reste der kürzeren Sequenz, die an dem Vergleich beteiligt ist und die ein "entsprechendes" Gegenstück in der anderen Sequenz besitzt, ermittelt. Für die Zwecke der vorliegenden Erfindung wird die Homologie dabei bevorzugt in üblicher Weise unter Verwendung der üblichen Algorithmen bestimmt. Erfindungsgemäß werden zum Vergleich nur die cDNAs der jeweiligen reifen Proteine herangezogen. Als homologe Sequenzen werden erfindungsgemäß ähnliche, bevorzugt identische, Sequenzgegenstücke mit Hilfe von bekannten Computerprogrammen ermittelt. Ein Beispiel für ein derartiges Programm ist das Programm Clone Manager Suite, das den Programmteil Align Plus enthält und von Scientific & Educational Software, Durham, NC, USA vertrieben wird. Dabei wird ein Vergleich zweier wie vorstehend definierter DNA-Sequenzen durchgeführt, unter der Option local alignment entweder nach der Methode FastScan - MaxScore oder nach der Methode Needleman-Wunsch unter Beibehaltung der Defaultwerte. Speziell wurde erfindungsgemäß zur Berechnung der Homologie die Programmversion "Clone Manager 7 Align Plus 5" mit den Funktionen "Compare Two Sequences/Local Fast Scan-Max Score/Compare DNA sequences" angewendet. Dabei wurden die aus den folgenden Quellen zugänglichen Algorithmen verwendet:
Die Erfindung betrifft ferner auch DNA-Sequenzen, die aufgrund der Degeneriertheit des genetischen Codes mit den erfindungsgemäßen Sequenzen verwandt sind sowie allelische Varianten davon. Die Degeneriertheit des genetischen Codes kann sich dabei aufgrund natürlicher Degeneriertheit oder aufgrund eines speziell gewählten Codongebrauchs ergeben. Natürlich vorkommende allelische Varianten können unter Verwendung gut bekannter Techniken der Molekularbiologie wie z. B. der Polymerase-Kettenreaktion (PCR) und Hybridisierungstechniken identifiziert werden.The invention further relates to DNA sequences which are related due to the degeneracy of the genetic code with the sequences of the invention as well as allelic variants thereof. The degeneracy of the genetic code may be due to natural degeneration or due to a specially chosen codon usage. Naturally occurring allelic variants may be synthesized using well-known molecular biology techniques, such as. The polymerase chain reaction (PCR) and hybridization techniques.
Eine DNA-Sequenz, die ein erfindungsgemäßes Polypeptid codiert, kann verwendet werden, um beliebige Wirtszellen wie beispielsweise Zellen von Pilzen, Hefen, Bakterien, Pflanzen oder Säugern zu transformieren. Derart transformierte Zellen zeichnen sich durch eine Produktion und ev. Sekretion an Phytase mit erhöhter Thermostabilität aus. Das so produzierte Phytaseenzym mit erhöhter Thermostabilität führt auch zu einer effizienten Phosphatfreisetzung aus Phytaten.A DNA sequence encoding a polypeptide of the invention can be used to transform any host cells, such as fungi, yeast, bacteria, plants or mammalian cells. Such transformed cells are characterized by a production and ev. Secretion of phytase with increased thermostability. The thus produced phytase enzyme with increased thermostability also leads to an efficient phosphate release from phytates.
Die Ausdrücke Protein, Peptid und Polypeptid sollen austauschbar verwendet werden. Ein Polypeptid oder Enzym mit Phytaseaktivität oder eine Phytase soll jedes Enzym bezeichnen, das die Freisetzung von anorganischem Phosphat aus verschiedenen Myoinositol-Phosphaten bewirken kann. Beispiele für solche Myoinositol-Phosphat(Phytase)-Substrate sind Phytinsäure und beliebige Salze davon, beispielsweise Natriumphytat oder Kaliumphytat oder gemischte Salze. Auch beliebige Positionsisomere der Di-, Tri-, Tetra- oder Pentaphosphate von Myoinositol können als Phytasesubstrat dienen. Die Phytaseaktivität kann unter Verwendung jedes beliebigen Assays, bei dem eines dieser Substrate verwendet wird, bestimmt werden. Eine erfindungsgemäße Phytase-Variante umfasst Polypeptidvarianten, die von einer speziellen Phytase durch Deletion oder Addition einer oder mehrerer Aminosäuren an das N-terminale und/oder C-terminale Ende des nativen Proteins, Deletion oder Addition einer oder mehrerer Aminosäuren an einer oder mehreren Stellen in dem nativen Protein oder Substitution einer oder mehrerer Aminosäuren an einer oder mehreren Stellen in der Phytase abgeleitet sind. Die Erzeugung solcher Varianten ist allgemein auf dem Fachgebiet bekannt. Beispielsweise können Aminosäuresequenz-Varianten der Polypeptide durch Mutation in der DNA hergestellt werden. Verfahren zur Mutagenese und Nucleotidsequenz-Veränderung sind auf dem Fachgebiet gut bekannt (vgl. beispielsweise
Derartige innerhalb einer Gruppe austauschbare Aminosäuren sind, ohne dadurch beschränkt zu werden, in der nachstehenden Tabelle beispielhaft angeführt.
Die Erfindung betrifft auch isolierte oder im Wesentlichen gereinigte Nucleinsäure- oder Proteinzusammensetzungen. Dabei bezeichnet ein isoliertes und gereinigtes Polynucleotid/Polypeptid bzw. Segment davon ein Polynucleotid bzw. Polypeptid bzw. Segment davon, das isoliert aus seiner nativen Umgebung vorliegt. Ein isoliertes Polynucleinsäuresegment oder Polypeptid kann in gereinigter Form vorliegen oder kann in einer nicht nativen Umgebung vorliegen, wie beispielsweise in einer transgenen Wirtszelle. Beispielsweise ist ein isoliertes oder gereinigtes Polynucleotidsegment oder -Protein oder ein biologisch aktiver Teil davon im Wesentlichen frei von weiterem zellulärem Material oder Kulturmedium bei Produktion nach rekombinanten Techniken oder im Wesentlichen frei von chemischen Vorläufern oder anderen chemischen Verbindungen. Bevorzugt ist ein isoliertes Polynucleotid frei von Sequenzen (bevorzugt Proteincodierenden Sequenzen), die natürlicherweise die Nucleinsäure (d.h. Sequenzen, die an den 5'- und 3'-Enden der Nucleinsäure lokalisiert sind) in der genomischen DNA des Organismus, aus dem die Nucleinsäure stammt, flankieren. Beispielsweise kann gemäß verschiedenen Ausführungsformen das isolierte Nucleinsäuremolekül weniger als etwa 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0,5 kb oder 0,1 kb Nucleotidsequenzen enthalten, die natürlicherweise das Nucleotinsäuremolekül in der genomischen DNA der Zelle, aus der die Nucleinsäure abgeleitet ist, flankieren. Ein Protein, das im Wesentlichen frei von zellulärem Material ist, umfasst Zusammensetzungen von Protein oder Polypeptid mit weniger als etwa 70%, 50%, 30%, 20%, 10%, 5% (bezogen auf das Trockengewicht) von verunreinigendem Protein. Wenn das erfindungsgemäße Protein oder ein biologisch aktives Fragment davon rekombinant produziert wird, umfasst bevorzugt das Kulturmedium weniger als etwa 70%, 50%, 30%, 20%, 10% oder 5% (bezogen auf das Trockengewicht) der chemischen Vorläufer oder nichtproteinartigen chemischen Substanzen. Fragmente und Varianten der erfindungsgemäßen Nucleotidsequenzen oder Proteine oder Proteinsegmente, die dadurch codiert werden, sind ebenfalls erfindungsgemäß umfasst. Fragment bezeichnet einen Teil der Nucleotidsequenz oder einen Teil der Aminosäuresequenz und daher einen Teil des Polypeptids oder Proteins, welches davon codiert wird.The invention also relates to isolated or substantially purified nucleic acid or protein compositions. An isolated and purified polynucleotide / polypeptide or segment thereof refers to a polynucleotide or polypeptide or segment thereof that is isolated from its native environment. An isolated polynucleic acid segment or polypeptide may be in purified form or may be in a non-native environment, such as in a transgenic host cell. For example, an isolated or purified polynucleotide segment or protein or biologically active portion thereof is substantially free of further cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemical compounds. Preferably, an isolated polynucleotide is free of sequences (preferably protein coding sequences) that naturally contain the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived flank. For example, according to various embodiments, the isolated nucleic acid molecule may comprise less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb nucleotide sequences that naturally comprise the nucleotonic acid molecule in the genomic DNA of the cell flanking the nucleic acid derived. A protein that is substantially free of cellular material includes protein or polypeptide compositions having less than about 70%, 50%, 30%, 20%, 10%, 5% (by dry weight) of contaminating protein. When the protein of the invention or a biologically active fragment thereof is produced recombinantly, preferably the culture medium comprises less than about 70%, 50%, 30%, 20%, 10% or 5% (by dry weight) of the chemical precursors or non-proteinaceous chemicals substances. Fragments and variants of the nucleotide sequences of the invention or proteins or protein segments encoded thereby are also included in the invention. Fragment refers to part of the nucleotide sequence or part of the amino acid sequence and therefore a portion of the polypeptide or protein encoded thereby.
Die Erfindung betrifft auch Expressionskassetten, die zur Einschleusung des eine erfindungsgemäße Phytase codierenden offenen Leserasters in eine Wirtszelle verwendet werden können. Sie umfassen bevorzugt eine Transkriptionsstartregion, die mit dem offenen Leseraster verknüpft ist. Eine derartige Expressionskassette kann eine Vielzahl von Restriktionsspaltstellen zur Insertion des offenen Leserasters und/oder anderer DNAs, z. B. einer Transkriptions-Regulator-Region und/oder selektierbare Markergene enthalten. Die Transkriptionskassette umfasst in der 5'→3'-Richtung der Transkription eine Transkriptions- und Translationsstartregion, die DNA-Sequenz von Interesse und eine Transkriptions- und Translationsstopregion, die in einer mikrobiellen Zelle funktionell ist. Die Terminationsregion kann bezüglich der Transkriptioninitiationsregion nativ sein, kann bezüglich der DNA-Sequenz von Interesse nativ sein oder kann von einer beliebigen anderen Quelle abgeleitet sein.The invention also relates to expression cassettes which can be used to introduce the phytase-encoding open reading frame into a host cell. They preferably comprise a transcription initiation region linked to the open reading frame. Such an expression cassette may have a plurality of restriction sites for insertion of the open reading frame and / or other DNAs, e.g. A transcriptional regulatory region and / or selectable marker genes. The transcriptional cassette in the 5 '→ 3' direction of transcription comprises a transcriptional and translational initiation region, the DNA sequence of interest, and a transcriptional and translational stop region that is functional in a microbial cell. The termination region may be native to the transcription initiation region, may be native to the DNA sequence of interest, or may be derived from any other source.
Der Ausdruck "offenes Leseraster" (ORF) bezeichnet die Aminosäure-Sequenz, die zwischen den Translationsstart- und -Stop-Codons einer codierenden Sequenz codiert wird. Die Ausdrücke "Start-Codon" und "Stop-Codon" bezeichnen eine Einheit aus drei benachbarten Nukleotiden (Codons) in einer codierenden Sequenz, die den Kettenstart und -stop der Proteinsynthese (mRNA-Translation) spezifizieren.The term "open reading frame" (ORF) refers to the amino acid sequence encoded between the translation start and stop codons of a coding sequence. The terms "start codon" and "stop codon" refer to a unit of three contiguous nucleotides (codons) in a coding sequence that specify the chain start and stop of protein synthesis (mRNA translation).
"Funktionelle Verknüpfung" bezeichnet im Zusammenhang mit einer Nukleinsäure eine Verbindung als Teil des gleichen Nukleinsäuremoleküls in geeigneter Positionierung und Orientierung zum Transkriptionsstart des Promotors. DNA in funktioneller Verknüpfung an einen Promotor befindet sich unter der Transkriptions-Initiations-Regulation des Promotors. Codierende Sequenzen können funktionell mit der Regulatorsequenz in Sense- oder Antisense-Orientierung verknüpft sein. Bei Bezugnahme auf Polypeptide bedeutet funktionelle Verknüpfung die Verbindung als Teil desselben Polypeptids, d.h. über Peptidylbindungen."Functional linkage" in the context of a nucleic acid refers to a compound as part of the same nucleic acid molecule in proper positioning and orientation for the transcription initiation of the promoter. DNA operably linked to a promoter is under the transcriptional initiation regulation of the promoter. Coding sequences may be operably linked to the regulatory sequence in sense or antisense orientation. When referring to polypeptides functional means Link the compound as part of the same polypeptide, ie via peptidyl bonds.
Erfindungsgemäß können beliebige Promotoren verwendet werden. Promotor bezeichnet die Nukleotidsequenz üblicherweise stromaufwärts (5') bezüglich der codierenden Sequenz und kontrolliert die Expression der codierenden Sequenz, indem die Erkennung für die RNA-Polymerase und anderer Faktoren, die für die richtige Transkription erforderlich sind, bereit gestellt wird. Der erfindungsgemäß verwendete Promotor kann einen Minimalpromotor umfassen, d.h. eine kurze DNA-Sequenz aus einer TATA-Box und anderen Sequenzen, die die Transkriptionsstartstelle spezifizieren, an die Regulatorelemente zur Kontrolle der Expression angefügt sind.Any promoters can be used according to the invention. Promoter usually refers to the nucleotide sequence upstream (5 ') to the coding sequence and controls the expression of the coding sequence by providing recognition for the RNA polymerase and other factors required for proper transcription. The promoter used in the invention may comprise a minimal promoter, i. a short DNA sequence from a TATA box and other sequences specifying the transcription start site to which the expression control elements are attached.
Der erfindungsgemäße Promotor kann auch eine Nucleotidsequenz umfassen, die einen Minimalpromotor und Regulatorelemente umfasst, der die Expression einer codierenden Sequenz oder funktionellen RNA kontrollieren kann. Dieser Typ von Promotorsequenz besteht aus proximalen und distalen stromaufwärts gelegenen Elementen, wobei die zuletzt genannten Elemente oft als Enhancer bezeichnet werden. Folglich ist ein Enhancer eine DNA-Sequenz, die die Promotor-Aktivität stimulieren kann und kann ein dem Promotor innewohnendes Element oder ein insertiertes heterologes Element sein, um die Expressionshöhe oder Gewebspezifität eines Promotors zu verstärken. Er kann in beiden Orientierungen funktionieren und kann selbst bei stromaufwärtiger oder stromabwärtiger Platzierung von dem Promotor funktionieren. Sowohl Enhancer als auch andere stromaufwärts gelegene Promotor-Elemente binden sequenzspezifisch DNA-bindende Proteine, die ihre Wirkungen vermitteln. Promotoren können in ihrer Gesamtheit von einem nativen Gen abgeleitet sein oder können aus verschiedenen Elementen zusammengesetzt sein, die von verschiedenen natürlich vorkommenden Promotoren abgeleitet sind oder können sogar aus synthetischen DNA-Segmenten zusammengesetzt sein. Ein Promotor kann auch DNA-Sequenzen enthalten, die an der Bindung von Proteinfaktoren beteiligt sind, die die Effizienz der Transkriptionsinitiation als Antwort auf physiologische oder entwicklungsbedingte Bedingungen kontrollieren.The promoter of the invention may also comprise a nucleotide sequence comprising a minimal promoter and regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and distal upstream elements, the latter elements often being referred to as enhancers. Thus, an enhancer is a DNA sequence that can stimulate promoter activity and may be an element inherent in the promoter or an inserted heterologous element to enhance the level of expression or tissue specificity of a promoter. It can function in both orientations and can function even on upstream or downstream placement of the promoter. Both enhancers and other upstream promoter elements sequence-specifically bind DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or may be composed of various elements derived from various naturally occurring promoters, or may even be composed of synthetic DNA segments. A promoter may also contain DNA sequences that participate in the binding of protein factors which control the efficiency of transcription initiation in response to physiological or developmental conditions.
Promotorelemente, insbesondere TATA-Elemente, die inaktiv sind oder eine stark verminderte Promotor-Aktivität in Abwesenheit einer stromaufwärtigen Aktivierung besitzen, werden als Minimalpromotoren oder Kernpromotoren bezeichnet. In Gegenwart eines geeigneten Transkriptionfaktors bzw. geeigneter Transkriptionsfaktoren liegt die Funktion des Minimalpromotors in der Ermöglichung der Transkription. Ein Minimal- oder Kernpromotor besteht somit nur aus allen Grundelementen, die für die Transkriptionsinitiation notwendig sind, z. B. einer TATA-Box und/oder einem Initiator.Promoter elements, especially TATA elements, which are inactive or have greatly reduced promoter activity in the absence of upstream activation are termed minimal promoters or core promoters. In the presence of a suitable transcription factor or transcription factors, the function of the minimal promoter is to allow transcription. A minimal or core promoter thus consists only of all basic elements necessary for transcription initiation, e.g. As a TATA box and / or an initiator.
Die Erfindung betrifft auch die erfindungsgemäße DNA enthaltende Vektoren. Diese Vektoren umfassen beliebige Plasmide, Cosmide, Phagen und andere Vektoren in doppelsträngiger oder einzelsträngiger, linearer oder zirkulärer Form, die gegebenenfalls selbst transmittierbar oder mobilisierbar sein können und die einen prokaryotischen oder eukaryotischen Wirt entweder durch Integration in das zelluläre Genom transformieren können oder die extrachromosomal vorliegen (z. B. autonom replizierende Plasmide mit einem Replikationsorigin).The invention also relates to the DNA-containing vectors of the invention. These vectors include any of plasmids, cosmids, phage and other vectors in double-stranded or single-stranded, linear or circular form which may optionally be self-transmissive or mobilizable and which may transform a prokaryotic or eukaryotic host either integrally into the cellular genome or which are extra-chromosomally present (eg, autonomously replicating plasmids with a replication origin).
Vektoren, Plasmide, Cosmide, künstliche Hefechromosomen (YACs), künstliche Bakterienchromosomen (BACs) und DNA-Segmente zur Verwendung zur Transformation von Zellen umfassen allgemein die erfindungsgemäße Phytase-codierende DNA sowie eine andere DNA, wie cDNA, ein Gen oder Gene, die in die Zellen eingeschleust werden sollen. Diese DNA-Konstrukte können weitere Strukturen wie Promotoren, Enhancer, Polylinker oder auch Regulatorgene, so weit erforderlich, umfassen. Eines der DNA-Segmente oder Gene, das bzw. die für die zelluläre Einschleusung ausgewählt wurde bzw. wurden, codiert/codieren zweckdienlicherweise ein Protein, das in den so erhaltenen transformierten (rekombinanten Zellen) exprimiert wird, was zu einem screenbaren oder selektierbaren Merkmal führt und/oder der transformierten Zelle einen verbesserten Phenotyp verleiht.Vectors, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and DNA segments for use in transforming cells generally include the phytase-encoding DNA of the present invention, as well as other DNA such as cDNA, a gene, or genes found in the cells are to be introduced. These DNA constructs may include other structures such as promoters, enhancers, polylinkers or regulatory genes, as necessary. One of the DNA segments or genes selected for cellular delivery suitably encodes / encodes a protein that is expressed in the transformed (recombinant) cells thus obtained, resulting in a screenable or selectable Feature and / or confer an improved phenotype on the transformed cell.
Die Konstruktion von Vektoren, die erfindungsgemäß verwendet werden können, ist einem Fachmann auf dem Gebiet angesichts der vorstehenden Offenbarung bekannt (vgl. z. B.
Die Vektoren können bereits Regulatorelemente enthalten, z. B. Promotoren, oder die erfindungsgemäßen DNA-Sequenzen können so manipuliert werden, dass sie solche Elemente enthalten. Geeignete Promotorelemente, die verwendet werden können, sind auf dem Fachgebiet bekannt und sind beispielsweise für Trichoderma reesei der cbh 1- oder der cbh-2-Promotor, für Aspergillus oryzae der amy-Promotor, für Aspergillus niger der xyl-, glaA-, alcA-, aphA-, tpiA-, gpdA-, sucl- und der pkiA-Promotor. Geeignete Promotorelemente, die zur Expression in Hefe verwendet werden können, sind auf dem Fachgebiet bekannt und sind beispielsweise der pho5-Promotor oder der gap-Promotor zur Expression in Saccharomyces cerevisiae und für Pichia pastoris, z. B. aoxl-Promotor oder der fmd-Promotor oder der mox-Promotor für H. polymorpha. The vectors may already contain regulatory elements, e.g. Promoters, or the DNA sequences of the invention can be manipulated to contain such elements. Suitable promoter elements which can be used are known in the art and are for example Trichoderma reesei the
DNA, die zur Einschleusung in Zellen geeignet ist, kann neben der erfindungsgemäßen DNA auch DNA, die aus einer beliebigen Quelle abgeleitet wurde oder daraus isoliert ist, umfassen. Ein Beispiel für eine abgeleitete DNA ist eine DNA-Sequenz, die als nützliches Fragment in einem gegebenen Organismus identifiziert wurde und die dann in im Wesentlichen reiner Form chemisch synthetisiert wurde. Ein Beispiel für eine solche DNA ist eine geeignete DNA-Sequenz, die beispielsweise unter Verwendung von Restriktionsendonucleasen erhalten wurde, so dass sie weiter erfindungsgemäß manipuliert werden kann, beispielsweise amplifiziert werden kann. Eine derartige DNA wird üblicherweise als rekombinante DNA bezeichnet. Daher umfasst eine geeignete DNA vollständig synthetische DNA, semisynthetische DNA, aus biologischen Quellen isolierte DNA und von eingeschleuster RNA abgeleitete DNA. Im Allgemeinen ist die eingeschleuste DNA kein ursprünglicher Bestandteil des Genotyps der Empfänger-DNA, aber erfindungsgemäß kann auch ein Gen aus einem gegebenen Genotyp isoliert und eventuell verändert werden und anschließend können Mehrfachkopien des Gens in den gleichen Genotyp eingeschleust werden, z. B. um die Produktion eines gegebenen Genprodukts zu verstärken.DNA suitable for introduction into cells, in addition to the DNA of the invention, may also comprise DNA derived from or isolated from any source. An example of a deduced DNA is a DNA sequence which has been identified as a useful fragment in a given organism and which has then been chemically synthesized in substantially pure form. An example of such a DNA is a suitable DNA sequence obtained, for example, using restriction endonucleases so that it can be further manipulated according to the invention, for example amplified. Such DNA is commonly referred to as recombinant DNA. Thus, a suitable DNA comprises wholly synthetic DNA, semisynthetic DNA, DNA isolated from biological sources, and DNA derived from in-blood RNA. In general, the introduced DNA is not an original part of the genotype of the recipient DNA, but according to the invention a gene from a given genotype can also be isolated and eventually altered and subsequently multiple copies of the gene can be introduced into the same genotype, e.g. To enhance production of a given gene product.
Die eingeschleuste DNA umfasst ohne Beschränkung DNA aus Genen, wie beispielsweise aus Bakterien, Hefen, Pilzen oder Viren. Die eingeschleuste DNA kann modifizierte oder synthetische Gene, Teile von Genen oder chimäre Gene einschließlich Genen aus dem gleichen oder einem unterschiedlichen Genotyp umfassen.The introduced DNA includes, without limitation, DNA from genes such as bacteria, yeasts, fungi or viruses. The introduced DNA may comprise modified or synthetic genes, parts of genes or chimeric genes, including genes from the same or a different genotype.
Die zur Transformation erfindungsgemäß verwendete DNA kann zirkulär oder linear, doppelsträngig oder einzelsträngig sein. Im Allgemeinen liegt die DNA in Form einer chimären DNA wie eine Plasmid-DNA vor, die auch codierende Regionen, die von Regulatorsequenzen flankiert sind, die die Expression der in der transformierten Zelle vorhandenen rekombinanten DNA unterstützen, enthalten. Beispielsweise kann die DNA selbst einen Promotor enthalten oder daraus bestehen, der in einer Zelle aktiv ist, der von einer Quelle, die sich von der Zelle unterscheidet, abgeleitet ist oder es kann ein Promotor verwendet werden, der bereits in der Zelle, d.h. der Transformationszielzelle, vorhanden ist.The DNA used for the transformation according to the invention may be circular or linear, double-stranded or single-stranded. In general, the DNA is in the form of a chimeric DNA, such as plasmid DNA, which also contains coding regions flanked by regulatory sequences that aid in the expression of the recombinant DNA present in the transformed cell. For example, the DNA itself may contain or consist of a promoter that is active in a cell that is from a source other than the cell is differentiated, derived or it can be used a promoter that is already present in the cell, ie the transformation target cell.
Im Allgemeinen ist die eingeschleuste DNA relativ klein, weniger als etwa 30 kb, um die Empfindlichkeit gegenüber physikalischem, chemischem oder enzymatischem Abbau zu minimieren, der mit der Größe der DNA zunimmt.In general, the introduced DNA is relatively small, less than about 30 kb, to minimize sensitivity to physical, chemical or enzymatic degradation, which increases with the size of the DNA.
Die Selektion eines geeigneten Expressionsvektors hängt von den Wirtszellen ab. Hefe- oder Pilzexpressionsvektoren können einen Replikationsorigin, einen geeigneten Promotor und Enhancer umfassen, und auch beliebige notwendige Ribosomenbindungsstellen, Polyadenylierungsstellen, Splice-Donor und -Akzeptor-Stellen, Transkriptionsterminationssequenzen und nicht-transkribierte 5'-flankierende Sequenzen umfassen.The selection of a suitable expression vector depends on the host cells. Yeast or fungal expression vectors may include a replication origin, a suitable promoter and enhancer, and also include any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed 5 'flanking sequences.
Beispiele für geeignete Wirtszellen sind: Pilzzellen der Gattung Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium, etc., wie beispielsweise Hefen der Gattungen Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula, Pichia und dergleichen. Geeignete Wirtssysteme sind beispielsweise Pilze wie Aspergilli, z. B. Aspergillus niger (ATCC 9142) oder Aspergillus ficuum (NRLL 3135) oder Trichoderma (z. B. Trichoderma reseei QM6a) und Hefen wie Saccharomyces, z. B. Saccharomyces cerevisiae oder Pichia, wie z. B. Pichia pastoris oder Hansenula, z. B. H. polymorpha (DSMZ 70277). Derartige Mikroorganismen können von anerkannten Hinterlegungsstellen, z. B. der American Type Culture Collection (ATCC), dem Centraalbureau voor Schimmelcultures (CBS) oder der Deutschen Sammlung für Mikroorganismen und Zellkulturen GmbH (DSMZ) oder beliebigen anderen Hinterlegungsstellen erhalten werden.Examples of suitable host cells are: fungal cells of the genus Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium, etc., such as yeasts of the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula, Pichia and the like. Suitable host systems are, for example, fungi such as Aspergilli, z. Aspergillus niger (ATCC 9142) or Aspergillus ficuum (NRLL 3135) or Trichoderma (eg Trichoderma reseei QM6a) and yeasts such as Saccharomyces, e.g. B. Saccharomyces cerevisiae or Pichia, such as. B. Pichia pastoris or Hansenula, z. B. H. polymorpha (DSMZ 70277). Such microorganisms may be used by recognized depositories, e.g. For example, the American Type Culture Collection (ATCC), the Centraalbureau voor Schimmelcultures (CBS) or the German Collection for Microorganisms and Cell Cultures GmbH (DSMZ) or any other depositories.
Die Expressionskassette kann in der 5'-3'-Transkriptionsrichtung eine Transkriptions- und Translationstartregion des erfindungsgemäßen Polynucleotids und eine Transkriptions- und Terminationsregion, die in vivo oder in vitro funktionell ist, enthalten. Die Terminationsregion kann bezüglich der Transkriptionsinitiationsregion nativ sein oder kann bezüglich des Polynucleotids nativ oder anderer Herkunft sein. Die Regulatorsequenzen können stromaufwärts (5' nichtcodierende Sequenzen), innerhalb (Intron) oder stromabwärts (3' nichtcodierende Sequenzen) einer codierenden Sequenz lokalisiert sein und die Transkription, das RNA-Processing oder die Stabilität und/oder die Translation der assoziierten codierenden Sequenz beeinflussen. Regulatorsequenzen können ohne Beschränkung Enhancer, Promotoren, Repressorbindungsstellen, Translationsleadersequenzen, Introns oder Polyadenylierungsignalsequenzen umfassen. Sie können natürliche und synthetische Sequenzen sowie Sequenzen umfassen, die eine Kombination aus synthetischen und natürlichen Sequenzen sind.The expression cassette may contain, in the 5'-3 'direction of transcription, a transcriptional and translational initiation region of the polynucleotide of the present invention and a transcription and termination region that is functional in vivo or in vitro. The termination region may be native to the transcription initiation region or may be native or polynucleotide related be of another origin. The regulatory sequences may be located upstream (5 'noncoding sequences), within (intron) or downstream (3' noncoding sequences) of a coding sequence, and affect transcription, RNA processing or stability and / or translation of the associated coding sequence. Regulator sequences may include, without limitation, enhancers, promoters, repressor binding sites, translational leader sequences, introns, or polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences that are a combination of synthetic and natural sequences.
Der erfindungsgemäß verwendete Vektor kann auch geeignete Sequenzen zur Amplifikation der Expression umfassen.The vector used in the invention may also comprise suitable sequences for amplification of expression.
Beispiele für Promotoren, die erfindungsgemäß verwendet werden können, sind Promotoren, von denen bekannt ist, dass sie die Expression in den eukaryotischen Zellen kontrollieren. Beliebige Promotoren mit der Fähigkeit zur Expression in Fadenpilzen können verwendet werden. Beispiele sind ein Promotor, der stark durch Stärke oder Zellulose induziert wird, z. B. ein Promotor für Glucoamylase oder α-Amylase aus der Gattung Aspergillus oder Cellulase (Cellobiohydrolase) aus der Gattung Trichoderma, ein Promotor für Enzyme im glykolytischen Stoffwechselweg, wie beispielsweise Phosphoglyceratkinase (PGK) und Glycerinaldehyd-3-phosphat-dehydrogenase (GPD), etc. Bevorzugt ist der Cellobiohydrolase-I-, der Cellobiohydrolase-II-, der Amylase-, der Glucoamylase-, der Xylanase- oder der Enolase-Promotor.Examples of promoters which can be used in the present invention are promoters known to control expression in the eukaryotic cells. Any promoters capable of expression in filamentous fungi can be used. Examples are a promoter which is strongly induced by starch or cellulose, e.g. A promoter for glucoamylase or α-amylase from the genus Aspergillus or cellulase (cellobiohydrolase) from the genus Trichoderma, a promoter for enzymes in the glycolytic pathway, such as phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPD), etc. Preferred is the cellobiohydrolase I, the cellobiohydrolase II, the amylase, the glucoamylase, the xylanase or the enolase promoter.
Zwei Hauptmethoden zur Kontrolle der Expression sind bekannt, d.h. Überexpression und Unterexpression. Überexpression kann durch Insertion einer oder mehrerer Extra-Kopien des ausgewählten Gens erzielt werden. Für die Unterexpression gibt es zwei Hauptmethoden, die üblicherweise auf dem Fachgebiet als "Antisense Downregulation" und "Sense Downregulation" bezeichnet werden. Im Allgemeinen werden diese Verfahren als "Gene Silencing" bezeichnet. Beide dieser Methoden führen zu einer Hemmung der Expression des Targetgens.Two major methods of controlling expression are known, ie overexpression and underexpression. Overexpression can be achieved by insertion of one or more extra copies of the selected gene. There are two main methods of underexpression commonly referred to in the art as "antisense downregulation" and "sense downregulation". In general, these methods are referred to as gene silencing. Both of these methods lead to an inhibition of the expression of the target gene.
Zusätzlich zur Verwendung eines speziellen Promotors können andere Typen von Elementen die Expression von Transgenen beeinflussen. Insbesondere wurde gezeigt, dass Introns das Potenzial zur Verstärkung der Transgenexpression besitzen.In addition to using a particular promoter, other types of elements can affect the expression of transgenes. In particular, introns have been shown to have the potential to enhance transgene expression.
Die Expressionskassette kann noch weitere Elemente umfassen, beispielsweise solche, die durch endogene oder exogene Elemente wie Zinkfinger-Proteine, einschließlich natürlich vorkommender Zinkfinger-Proteine oder chimärer Zinkfinger-Proteine, reguliert werden können.The expression cassette may include other elements, for example, those that may be regulated by endogenous or exogenous elements, such as zinc finger proteins, including naturally occurring zinc finger proteins or zinc protein chimeric proteins.
Die erfindungsgemäß verwendete Expressionskassette kann ferner Enhancer-Elemente oder stromaufwärtige Promotor-Elemente enthalten.The expression cassette used in the invention may further contain enhancer elements or upstream promoter elements.
Vektoren zur erfindungsgemäßen Verwendung können so konstruiert werden, dass sie ein Enhancer-Element enthalten. Die erfindungsgemäßen Konstrukte umfassen somit das Gen von Interesse zusammen mit einer 3'-DNA-Sequenz, die als Signal wirkt, um die Transkription zu terminieren und die Polyadenylierung der so erhaltenen mRNA zu erlauben. Es können beliebige Signalsequenzen verwendet werden, die die Sekretion aus dem gewählten Wirtsorganismus ermöglichen. Bevorzugte Signalsequenz ist die Phytase-Signalsequenz aus Aspergillus niger oder daraus abgeleitete Signalsequenzen für die Sekretion aus filamentösen Pilzen.Vectors for use in the invention can be engineered to contain an enhancer element. The constructs of the invention thus comprise the gene of interest together with a 3 'DNA sequence which acts as a signal to terminate transcription and allow polyadenylation of the mRNA thus obtained. Any signal sequences that allow secretion from the chosen host organism can be used. Preferred signal sequence is the phytase signal sequence from Aspergillus niger or derived therefrom signal sequences for secretion from filamentous fungi.
Es kann auch eine spezielle Leadersequenz verwendet werden, da die DNA-Sequenz zwischen der Transkriptionsstartstelle und dem Start der codierenden Sequenz, d.h. der nicht-translatierten Leadersequenz die Genexpression beeinflussen kann. Bevorzugte Leadersequenzen umfassen Sequenzen, die die optimale Expression des angehefteten Gens steuern, d.h sie umfassen eine bevorzugte Consensus-Leader-Sequenz, die die mRNA-Stabilität erhöht oder erhält und eine ungeeignete Translationsinitiation verhindert. Die Wahl solcher Sequenzen ist einem Fachmann auf dem Gebiet gut bekannt.A specific leader sequence can also be used since the DNA sequence between the transcription start site and the start of the coding sequence, ie the untranslated leader sequence, can influence gene expression. Preferred leader sequences include sequences that direct the optimal expression of the attached gene, ie, they include a preferred consensus leader sequence that increases or maintains mRNA stability and prevents inappropriate translation initiation. The choice of such sequences is well known to one skilled in the art.
Um die Möglichkeit, die Transformanten zu identifizieren, zu verbessern, kann ein selektierbares oder screenbares Markergen in die Expressionskassette aufgenommen werden. Derartige Markergene sind einem Fachmann auf dem Gebiet gut bekannt.To improve the ability to identify the transformants, a selectable or screenable marker gene can be incorporated into the expression cassette. Such marker genes are well known to one skilled in the art.
Die Expressionskassette oder ein Vektorkonstrukt, das die Expressionskassette enthält, wird in eine Wirtszelle eingeführt. Eine Vielzahl von Techniken sind verfügbar und einem Fachmann auf dem Gebiet zur Einschleusung von Konstrukten in eine Wirtszelle gut bekannt. Die Transformation mikrobieller Zellen kann unter Verwendung von Polyethylenglykol, Calciumchlorid, viraler Infektion, DEAE-Dextran, Phageninfektionen, Elektroporation und anderen auf dem Fachgebiet bekannten Methoden durchgeführt werden. Die Transformation von Pilzen kann nach
Sobald die erfindungsgemäße Expressionskassette bzw. DNA-Sequenz erhalten ist, kann sie in Vektoren nach an sich bekannten Verfahren eingefügt werden, um das codierte Polypeptid in geeigneten Wirtssystemen zu überexprimieren. Jedoch können auch DNA-Sequenzen als solche verwendet werden, um geeignete Wirtssysteme der Erfindung zu transformieren, um eine Überexpression des codierten Polypeptids zu erzielen.Once the expression cassette or DNA sequence of the present invention is obtained, it can be inserted into vectors by methods known per se in order to overexpress the encoded polypeptide in suitable host systems. However, DNA sequences as such can also be used to transform suitable host systems of the invention to achieve overexpression of the encoded polypeptide.
Sobald eine erfindungsgemäße DNA-Sequenz in einer geeigneten Wirtszelle in einem geeigneten Medium exprimiert wurde, kann die codierte Phytase entweder aus dem Medium, falls die Phytase in das Medium sezerniert wird, oder aus dem Wirtsorganismus, falls die Phytase intrazellulär z. B. im periplasmatischen Raum vorhanden ist, nach an sich bekannten Verfahren aufkonzentriert und/oder isoliert werden. Bekannte Verfahren der Abtrennung der unlöslichen Bestandteile des Kulturmediums und der Biomasse gefolgt von Verfahren zur Aufkonzentrierung der Phytase können zur Herstellung von konzentrierten Phytaselösungen oder als Vorbereitung zur Trocknung der Phytase angewendet werden. Beispielsweise können Filtrationsverfahren oder Zentrifugationsverfahren zur Abtrennung der unlöslichen Bestandteile verwendet werden gefolgt von Ultrafiltrationsverfahren zur Aufkonzentration, oder es werden Cross-flow-Filtrationsverfahren angewendet. Die Trocknung kann durch Gefrier- und Sprühtrocknen, Granulationsverfahren, Extrudierung oder andere Verfahren erfolgen. Bekannte Verfahren der Proteinreinigung können verwendet werden, um die erfindungsgemäßen Phytasen zu isolieren. Beispielsweise können verschiedene chromatographische oder gelchromatographische Verfahren einzeln oder in Kombination verwendet werden. In Abhängigkeit von der verwendeten Wirtszelle bei einem rekombinanten Produktionsverfahren kann das erfindungsgemäße Enzym kovalent durch Glykosylierung modifiziert sein oder nicht. In eukaryotischen Zellen dient die Glykosylierung der sezernierten Proteine dazu, die Proteinfaltung, die Konformationsstabilität, die thermische Stabilität und die Resistenz gegenüber Proteolyse zu modulieren. Im Hinblick auf eine spezifische Anwendung der Phytase kann eine glykosylierte Variante des Enzyms gegenüber einer nicht-glykosilierten Variante bevorzugt sein. Beispielsweise dient die Verwendung einer glykosylierten Phytase in Tierfutter zum Schutz des Enzyms vor thermischer Denaturierung während der Futterpelletisierung und vor proteolytischer Inaktivierung bei der Passage durch den Magen des Tieres, wodurch die Verteilung des aktiven Enzyms im Darmtrakt und an die Wirkstelle gefördert wird. Für Anwendungen in der Nahrungsmittelverarbeitung, bei der die Enzymaktivität nur während der Verarbeitung und nicht im Endprodukt erwünscht ist, kann eine thermolabile, d.h. nicht-glykosylierte, und gegenüber protolytischem Abbau empfindlichere Phytase bevorzugt sein.Once a DNA sequence of the invention has been expressed in a suitable host cell in a suitable medium, the encoded phytase may be secreted either from the medium if the phytase is secreted into the medium or from the host organism if the phytase is intracellularly e.g. B. is present in the periplasmic space, concentrated and / or isolated according to known methods. Known method of separating the insoluble Culture medium and biomass components followed by phytase concentration techniques may be used to prepare concentrated phytase solutions or to prepare the phytase to dry. For example, filtration methods or centrifugation methods may be used to separate the insoluble components, followed by ultrafiltration methods for concentration, or cross-flow filtration methods are used. Drying may be by freeze and spray drying, granulation, extrusion or other methods. Known methods of protein purification can be used to isolate the phytases of the invention. For example, various chromatographic or gel chromatographic methods can be used singly or in combination. Depending on the host cell used in a recombinant production process, the enzyme of the invention may or may not be covalently modified by glycosylation. In eukaryotic cells, glycosylation of the secreted proteins serves to modulate protein folding, conformational stability, thermal stability, and resistance to proteolysis. With regard to a specific application of the phytase, a glycosylated variant of the enzyme may be preferred over a non-glycosylated variant. For example, the use of a glycosylated phytase in animal feed to protect the enzyme from thermal denaturation during food pelleting and proteolytic inactivation upon passage through the animal's stomach promotes distribution of the active enzyme in the intestinal tract and to the site of action. For food processing applications where enzyme activity is desired only during processing and not in the final product, a thermolabile, ie, non-glycosylated, and more susceptible to protolytic degradation, may be preferred.
Die Erfindung betrifft auch Phytasezusammensetzungen, die das erfindungsgemäße Polypeptid enthalten. Im Allgemeinen sind Phytasezusammensetzungen flüssig oder trocken. Flüssige Zusammensetzungen enthalten das Phytaseenzym bevorzugt in einer gereinigten oder angereicherten Form. Jedoch können auch Hilfsstoffe wie beispielsweise ein Stabilisator wie Glycerin, Sorbit oder Monopropylenglykol, Additive wie Salze, Zucker, Konservierungsstoffe, Mittel zur Einstellung des pH-Werts, Proteine und Phytat oder Salze von Myoinositolphosphaten (ein Phytasesubstrat) zugesetzt werden. Typische Flüssigzusammensetzungen sind wässrige oder ölige Aufschlämmungen. Die Flüssigzusammensetzungen können einem Nahrungsmittel oder Futtermittel vor oder nach einer möglichen Pelletierung bzw. Verarbeitungsschritt zugesetzt werden.The invention also relates to phytase compositions containing the polypeptide of the invention. In general, phytase compositions are liquid or dry. Liquid compositions preferably contain the phytase enzyme in a purified or enriched form. however Also, adjuvants such as a stabilizer such as glycerol, sorbitol or monopropylene glycol, additives such as salts, sugars, preservatives, pH adjusters, proteins and phytate or salts of myoinositol phosphates (a phytase substrate) may be added. Typical liquid compositions are aqueous or oily slurries. The liquid compositions may be added to a food or feed before or after a possible pelleting or processing step.
Trockenzusammensetzungen können gefriergetrocknete, sprühgetrocknete, granulierte oder extrudierte Zusammensetzungen sein, die ausschließlich das Enzym enthalten können. Vor dem Trocknen können aber auch Stoffe zugesetzt werden um den pH-Wert zu regulieren, sowie weitere Additive und Füllstoffe wie Maltodextrine, Lactose, Magermilchpulver, Salze zweiwertiger Kationen wie die von Mg, Ca, Zn, etc.. Trockenzusammensetzungen können Granulate sein, die leicht beispielsweise mit Nahrungsmitteln oder Futterkomponenten gemischt werden können oder bevorzugter eine Komponente eines Prämix bilden. Die Teilchengröße des Enzymgranulats ist bevorzugt mit der der anderen Komponenten des Gemisches kompatibel. Dies ermöglicht sichere und zweckdienliche Mittel, um Enzyme beispielsweise in prozessierte Nahrungsmittel, Prämixe oder Tierfutter einzuarbeiten.Dry compositions may be freeze-dried, spray-dried, granulated or extruded compositions which may contain only the enzyme. However, before drying, substances may also be added to control the pH, as well as other additives and fillers such as maltodextrins, lactose, skimmed milk powder, salts of divalent cations such as Mg, Ca, Zn, etc. Dry compositions may be granules which may be easily mixed with, for example, food or feed components, or more preferably form a component of a premix. The particle size of the enzyme granules is preferably compatible with that of the other components of the mixture. This allows safe and convenient means to incorporate enzymes into, for example, processed foods, premixes or animal feed.
Beispielsweise kann eine stabile Formulierung des erfindungsgemäßen Phytaseenzyms dadurch hergestellt werden, dass ein Gemisch aus einer flüssigen Enzymlösung auf ein Füllmittel wie Maltodextrin gesprüht wird und dann das Gemisch getrocknet wird, oder der flüssigen Enzymlösung wird vor dem Trocknen 20%-60% Magermilchpulver (w/w bezogen auf Gesamtprotein der Enzymlösung) und/oder 1%-5% Calciumpropionat (w/w bezogen auf Gesamtprotein der Enzymlösung) zugesetzt und der pH-Wert auf einen definierten Wert eingestellt, insbesondere auf pH 5,2 ± 0,5. Die Verringerung der Feuchtigkeit und die Bindungswechselwirkungen der Phytase mit dem Füllmittel schützen das Enzym zusätzlich zu seiner in seiner Struktur definierten Stabilität vor Umwelteinflüssen wie Temperaturextrema, die während der Herstellung des Futtermittels auftreten können. Trockene und flüssige Formulierungen können weiter stabilisiert werden, wenn die Aktivität potenzieller proteolytischer Enzyme verringert wird, die als Nebenprodukte in dem Flüssigfermentationsgemisch vorhanden sein können, das zur Herstellung des erfindungsgemäßen Enzyms verwendet wird. Das so erhaltene Trockenenzym-Gemisch kann als Futterergänzungsmittel zur Verwendung in der Geflügel- und Schweinezucht verwendet werden. Ferner führt eine Verringerung der Phosphat-Supplementation zu einer Abnahme der Phosphat-Verunreinigung, die ihrerseits signifikant die Umweltbelastung durch intensive Tierzucht verringert.For example, a stable formulation of the phytase enzyme of the present invention can be prepared by spraying a mixture of a liquid enzyme solution onto a filler such as maltodextrin and then drying the mixture, or 20% -60% skimmed milk powder (w / o) before drying the liquid enzyme solution. w based on total protein of the enzyme solution) and / or 1% -5% calcium propionate (w / w based on total protein of the enzyme solution) was added and the pH was adjusted to a defined value, in particular to pH 5.2 ± 0.5. The reduction in moisture and the binding interactions of the phytase with the filler protect the enzyme, in addition to its stability defined in its structure, from environmental influences such as temperature extremes that occur during the manufacture of the feed may occur. Dry and liquid formulations can be further stabilized by reducing the activity of potential proteolytic enzymes which may be present as by-products in the liquid fermentation mixture used to prepare the enzyme of the present invention. The resulting dry enzyme mixture can be used as a feed supplement for use in poultry and pig breeding. Furthermore, a reduction in phosphate supplementation leads to a decrease in phosphate contamination, which in turn significantly reduces the environmental impact of intensive stock farming.
Sobald ein Trockenenzym-Präparat erhalten ist, kann daraus in weiteren Schritten ein Agglomerations-Granulat hergestellt werden. Hierfür wird ein Mischer mit hohen Scherkräften verwendet, wodurch Füllmaterial und das Enzym coagglomerieren und ein Granulat gebildet wird. Absorptionsgranulate werden hergestellt, indem Kerne aus einem Trägermaterial durch das erfindungsgemä-βe Enzym beschichtet werden. Typische Füllmaterialien sind Salze wie Dinatriumsulfat. Andere Füllmaterialien umfassen Kaolin, Talk, Magnesiumaluminiumsilicat und Cellulosefasern. Gegebenenfalls werden Bindemittel wie Dextrine auch in das Agglomerationsgranulat aufgenommen.As soon as a dry enzyme preparation is obtained, an agglomeration granulate can be produced in further steps. For this purpose, a high shear mixer is used which coagglomerates filler and the enzyme and granules are formed. Absorption granules are prepared by coating cores from a carrier material by the enzyme according to the invention. Typical fillers are salts, such as disodium sulfate. Other fillers include kaolin, talc, magnesium aluminum silicate, and cellulosic fibers. Optionally, binders such as dextrins are also incorporated in the agglomeration granules.
Typische Trägermaterialien umfassen Stärke, z. B. in Form von Cassava, Kartoffel und Getreide, insbesondere Mais, Reis und Weizen, oder proteinhaltige Erzeugnisse wie z. B. Sojaprotein. Salze können auch verwendet werden. Das Granulat wird gegebenenfalls mit einem Beschichtungsgemisch umhüllt. Ein solches Gemisch umfasst Beschichtungsmittel, bevorzugt hydrophobe Beschichtungsmittel, dehydriertes Palmöl und Talk und gegebenenfalls andere Additive wie Calciumcarbonat oder Kaolin um die Bioverfügbarkeit an der bestimmungsgemäßen Wirkstätte zu verbessern.Typical support materials include starch, e.g. B. in the form of cassava, potato and cereals, especially corn, rice and wheat, or proteinaceous products such. B. soy protein. Salts can also be used. The granules are optionally coated with a coating mixture. Such a mixture comprises coating agents, preferably hydrophobic coating agents, dehydrated palm oil and talc, and optionally other additives such as calcium carbonate or kaolin in order to improve the bioavailability at the intended site.
Zusätzlich können die Mischungen mit Phytase andere Stoffe wie farbgebende Mittel, Aromaverbindungen, Stabilisatoren, Vitamine, Mineralien, andere Nahrungsmittel- oder Futter-Enzyme und dergleichen enthalten. Dies trifft insbesondere für die sogenannten Prämixe zu.In addition, the mixtures with phytase may contain other substances such as coloring agents, aroma compounds, stabilizers, vitamins, minerals, other food or feed enzymes and the like. This is especially true for the so-called premixes.
Ein Nahrungsmittel- oder Futtermitteladditiv ist eine im Wesentlichen reine Verbindung oder eine Zusammensetzung aus mehreren Verbindungen, die dazu bestimmt oder geeignet ist, Nahrungsmitteln oder Futtermitteln zugesetzt zu werden. Insbesondere ist es eine Substanz, die gemäß ihrem Verwendungszweck eine Komponente eines Nahrungsmittels oder Futtermittels wird oder Eigenschaften eines Nahrungsmittel- oder Futtermittelprodukts beeinflusst. So soll ein Phytaseadditiv eine Phytase bezeichnen, die kein natürlicher Bestandteil der hauptsächlich für Nahrungsmittel und Futtermittel verwendeten Substanzen ist oder in ihrer natürlichen Konzentration darin nicht vorhanden ist. Beispielsweise wird die Phytase dem Futtermittel separat von den Futtermittelsubstanzen allein oder in Kombination mit anderen Futtermitteladditiven zugesetzt. Ein typischer Prämix umfasst üblicherweise eine oder mehrere Verbindungen wie Vitamine, Mineralien oder Futter-verstärkende Enzyme und geeignete Träger und/oder Exzipienzien.A food or feed additive is a substantially pure compound or compound of a number of compounds which is intended or suitable to be added to food or feed. In particular, it is a substance that becomes a component of a food or feed according to its purpose or influences properties of a food or feed product. Thus, a phytase additive is intended to refer to a phytase which is not a natural constituent of the substances principally used for food and feed or is absent in their natural concentration therein. For example, the phytase is added to the feed separately from the feeds alone or in combination with other feed additives. A typical premix will usually comprise one or more compounds such as vitamins, minerals or feed-enhancing enzymes, and suitable carriers and / or excipients.
Ein gebrauchsfertiges Phytaseadditiv ist ein Additiv, das nicht in situ in Tierfutter oder in verarbeiteten Nahrungsmitteln produziert wird. Ein gebrauchsfertiges Phytaseadditiv kann Menschen oder Tieren direkt oder bevorzugt direkt nach dem Mischen mit anderen Bestandteilen des Futters oder der Nahrungsmittel verabreicht werden. Beispielsweise wird ein Futteradditiv gemäß diesem Aspekt der vorliegenden Erfindung mit anderen Futtermitteln und -additiven unter Erhalt eines Prämixes oder Ergänzungsfutters vereinigt. Solche anderen Futtermittelbestandteile umfassen eine oder mehrere andere (bevorzugt thermostabile) Enzymsupplemente, andere Futteradditive, mineralische Futtermittel und Aminosäuren. Die so erhaltenen (kombinierten) Futteradditive können mehrere verschiedene Verbindungstypen umfassen und können dann in ihrer geeigneten Menge mit den Futtermitteln wie Getreide- und Proteinträgern unter Bildung eines zusammengesetzten Tierfutters vermischt werden. Die Verarbeitung dieser Komponenten zu einem Tierfutter kann nach dem Mischen mit an sich bekannten Verarbeitungsvorrichtungen wie einer Doppelpelletiermaschine, eines Dampfpelletierers, eines Expanders oder eines Extruders durchgeführt werden.A ready-to-use phytase additive is an additive that is not produced in situ in animal feed or in processed foods. A ready-to-use phytase additive may be administered to humans or animals directly or preferably directly after mixing with other ingredients of the feed or foods. For example, a feed additive according to this aspect of the present invention is combined with other feeds and additives to obtain a premix or supplemental feed. Such other feed ingredients include one or more other (preferably thermostable) enzyme supplements, other feed additives, mineral feeds and amino acids. The (combined) feed additives thus obtained may comprise several different types of compounds and may then be mixed in their appropriate amount with feeds such as cereal and protein carriers to form a composite animal feed. The processing of these components into an animal feed may be known per se after mixing Processing devices such as a Doppelpelletiermaschine, a Dampfpelletierers, an expander or an extruder are performed.
Auf ähnliche Weise kann ein Nahrungsmitteladditiv gemäß dieser Ausführungsform der vorliegenden Erfindung mit anderen Nahrungsmittelkomponenten vereinigt werden, wodurch verarbeitete Nahrungsmittelprodukte erzeugt werden. Solche anderen Nahrungsmittelkomponenten umfassen ein oder mehrere Enzymsupplemente, Vitamine, Mineralien und Spurenelemente. Das so erhaltene kombinierte Nahrungsergänzungsmittel kann dann in einer geeigneten Menge mit anderen Nahrungsmittel-Komponenten wie Getreide und Pflanzenproteine vermischt werden, um ein verarbeitetes Nahrungsmittel zu ergeben. Die Verarbeitung dieser Komponenten zu einem verarbeiteten Nahrungsmittel kann unter Verwendung an sich bekannter Verarbeitungsvorrichtungen durchgeführt werden.Similarly, a food additive according to this embodiment of the present invention may be combined with other food components to produce processed food products. Such other food components include one or more enzyme supplements, vitamins, minerals and trace elements. The combined dietary supplement thus obtained may then be mixed in an appropriate amount with other food components, such as cereals and vegetable proteins, to yield a processed food. The processing of these components into a processed foodstuff may be carried out using processing devices known per se.
In einer bevorzugten Ausführungsform umfassen die erfindungsgemäßen Phytasezusammensetzungen zusätzlich eine wirksame Menge eines oder mehrerer Enzyme für Nahrungs- oder Futtermittel, bevorzugt ausgewählt aus alpha-Galactosidasen, beta-Galactosidasen, Laccasen, anderen Phytasen, Phosphatasen, Endoglucanasen, insbesondere endo-beta-1,4-Glucanasen, endo-beta-1,3(4)-Glucanasen, endo-1,2-beta-Glucanasen und endo-1,3-alpha-Glu-canasen, Cellulasen, Xylosidasen, Galactanasen, insbesondere Arabinogalactan-endo-1,4-beta-Galactosidasen und Arabinogalactan-endo-1,3-beta-Galactosidasen, Pectin-abbauenden Enzymen, insbesondere Pectinasen, Pectinesterasen, Pectinlyasen, Polygalacturonasen, Arabananasen, Rhamnogalacturonasen, Rhamnogalacturonanacetylesterasen, Rhamnogalacturonan-alpha-Rhamnosidasen, Pectatlyasen und alpha-Galacturonidasen, Mannanasen, beta-Mannosidasen, Mannanacetylesterasen, Xylanacetylesterasen, Proteasen, Xylanasen, Arabinoxylanasen und lipolytischen Enzymen wie Lipasen, Phospholipasen und Cutinasen.In a preferred embodiment, the phytase compositions of the invention additionally comprise an effective amount of one or more enzymes for food or feed, preferably selected from alpha-galactosidases, beta-galactosidases, laccases, other phytases, phosphatases, endoglucanases, in particular endo-beta-1,4 Glucanases, endo-beta-1,3 (4) -glucanases, endo-1,2-beta-glucanases and endo-1,3-alpha-glucanases, cellulases, xylosidases, galactanases, especially arabinogalactan-endo-1 , 4-beta-galactosidases and arabinogalactan-endo-1,3-beta-galactosidases, pectin degrading enzymes, in particular pectinases, pectin esterases, pectin lyases, polygalacturonases, arabananases, rhamnogalacturonases, rhamnogalacturonanacetylesterases, rhamnogalacturonan-alpha-rhamnosidases, pectate lyases and alpha-galacturonidases , Mannanases, beta-mannosidases, mannanacetylesterases, xylanacetylesterases, proteases, xylanases, arabinoxylanases and lipolytic enzymes such as lipases, Pho spholipases and cutinases.
Das erfindungsgemäße Tierfutteradditiv wird dem Tier vor oder gleichzeitig mit dem Futter verabreicht. Bevorzugt wird das erfindungsgemäße Tierfutteradditiv dem Tier gleichzeitig mit dem Futter verabreicht.The animal feed additive according to the invention is administered to the animal before or simultaneously with the feed. The animal feed additive according to the invention is preferably administered to the animal simultaneously with the feed.
Eine effektive Menge an Phytase in Nahrungsmitteln oder Futtermitteln beträgt etwa 10-20.000 PPU/kg, bevorzugt etwa 10-15.000 PPU/kg, bevorzugter etwa 10-10.000 PPU/kg, noch bevorzugter etwa 50-5.000 PPU/kg, insbesondere 50-2.000 PPU/kg Futter oder Nahrungsmittel.An effective amount of phytase in food or feed is about 10-20,000 PPU / kg, preferably about 10-15,000 PPU / kg, more preferably about 10-10,000 PPU / kg, even more preferably about 50-5,000 PPU / kg, especially 50-2,000 PPU / kg feed or food.
Die Erfindung betrifft auch die Verwendung von Phytase zur Verarbeitung und Herstellung von Nahrungsmitteln und Futtermitteln. Getreide und Mehle für Nahrungsmittel können enzymatisch mit Phytase behandelt werden, um den Phytingehalt der Rohstoffe zu verringern. Verringerte Phytingehalte verbessern die Nahrungsmittelqualität, indem die Verfügbarkeit essenzieller Mineralien wie Eisen, Calcium und Zink erhöht wird. Zusätzlich zur Erhöhung der Qualität der Nahrungsmittel kann die Verwendung von Phytase während der Verarbeitung die Gesamteffizienz der Nahrungsmittelherstellung verbessern. Beispielsweise kann die Zugabe von Phytase zu weißen Sojabohnenflocken während der Herstellung eines Sojaproteinisolats signifikant die Ausbeute und Qualität des extrahierbaren Proteins erhöhen. Dabei ist die Phytase nur während der Herstellung und Verarbeitung aktiv und ist in dem Endprodukt nicht mehr aktiv. Dieser Aspekt ist insbesondere bei der Teigherstellung und beim Backen und der Produktion anderer verzehrfertiger Produkte auf Getreidebasis von Bedeutung. Auf ähnliche Weise können Tierfutterkomponenten wie getoastetes Sojabohnenmehl oder Canolamehl mit Phytase vor dem eigentlichen Herstellungsprozess vorbehandelt werden. Die Entfernung von anti-nutritiven Faktoren in Tierfutterkomponenten vor der Herstellung führt zu einer physiologisch höheren Qualität und zu wertvolleren Tierfutterinhaltsstoffen. Bei diesen Verarbeitungsverfahren ist die Phytase während der Herstellung aktiv und ist im Verdauungstrakt des Tiers nach Aufnahme des behandelten Futters in der Regel nicht mehr aktiv.The invention also relates to the use of phytase for the processing and production of food and feed. Food grains and flours can be enzymatically treated with phytase to reduce the phytin content of the raw materials. Reduced phytin levels improve food quality by increasing the availability of essential minerals such as iron, calcium and zinc. In addition to increasing the quality of foods, the use of phytase during processing can improve the overall efficiency of food production. For example, the addition of phytase to white soybean flakes during the production of a soy protein isolate can significantly increase the yield and quality of the extractable protein. The phytase is only active during production and processing and is no longer active in the final product. This aspect is particularly important in dough preparation and in the baking and production of other ready-to-eat cereal-based products. Similarly, animal feed components such as toasted soybean meal or canola meal with phytase may be pretreated prior to the actual manufacturing process. The removal of anti-nutritive factors in animal feed components prior to preparation results in a higher physiological quality and more valuable animal feed ingredients. In these processing methods, the phytase is active during production and is generally no longer active in the digestive tract of the animal after ingestion of the treated feed.
Zusätzlich zur Verwendung von Phytase als Futterverarbeitungshilfsmittel betrifft die vorliegende Erfindung die Verwendung der erfindungsgemäßen Phytase als Verdauungshilfe. Phytase in Tablettenform kann zusammen mit der Nahrungsaufnahme eingenommen werden, um das aktive Enzym im Magen-Darm-Trakt zu verteilen.In addition to the use of phytase as a feed processing aid, the present invention relates to the use of the phytase of the invention as a digestive aid. Phytase in tablet form can be taken with food intake to distribute the active enzyme in the gastrointestinal tract.
Die erfindungsgemäße Phytase kann ebenso in vorteilhafter Weise bei monogastrischen wie polygastrischen Tieren, insbesondere bei jungen Kälbern zum Einsatz kommen. Futter für Fische und Schalentiere kann ebenfalls mit Phytase supplementiert werden, um die Ausnutzung des Futters zu verbessern und den Gehalt an ausgeschiedenem Phosphor bei intensiver Tierzucht zu verringern. Das erfindungsgemäße Futtermittel kann auch Tieren wie Geflügel, z. B. Masthühnern, Truthühnern, Gänsen, Enten sowie Schweinen, Pferden, Rindern, Schafen, Ziegen, Hunden und Katzen sowie Fischen und Schalentieren verabreicht werden. Es ist jedoch besonders bevorzugt, dass das erfindungsgemäße Futtermittel Schweinen oder Geflügel verabreicht wird.The phytase according to the invention can also be advantageously used in monogastric and polygastric animals, in particular in young calves. Feed for fish and shellfish may also be supplemented with phytase to improve feed utilization and reduce levels of excreted phosphorus in intensive livestock. The feed of the invention may also animals such as poultry, z. As chickens, turkeys, geese, ducks and pigs, horses, cattle, sheep, goats, dogs and cats and fish and shellfish are administered. However, it is particularly preferred that the feed according to the invention is administered to pigs or poultry.
Erfindungsgemäße Phytaseformulierungen können auch mit anderen Inhaltsstoffen kombiniert werden, wodurch neue und besonders vorteilhafte Futterzusammensetzungen entstehen. Da, wie vorstehend ausgeführt, die Verfügbarkeit von pflanzlichem Phosphat in Sojabohnenmehl und Getreide in Folge der Bindung an Phytinsäure niedrig ist, wird anorganisches Phosphat dem Futter zugesetzt, um eine adäquate Phosphorversorgung der Tiere zu gewährleisten. Jedoch enthalten diese Futtermittel zu viel Gesamtphosphat und führen dadurch zu einer Verschmutzung der Umwelt mit Phosphat. Speziell umfasst das erfindungsgemäße Tierfuttermittel die Kombination einer erfindungsgemäßen Phytase mit Tierfutterinhaltsstoffen, um ein Futtermittel zu ergeben, das wesentlich erniedrigte Gehalte an zugesetztem anorganischem Phosphor enthält. In einer bevorzugten Ausführungsform umfasst das erfindungsgemäße Futtermittel typische Futterinhaltsstoffe, Mikronährstoffe, Spurenelemente, Vitamine, etc. sowie eine effektive Menge an Phytase und anorganischem Phosphor, wobei die Mengen der Phytase und des Phosphors zwischen 50 und 20.000 Einheiten Phytase/kg Futtermittel und weniger als 0,45% anorganischem Phosphor, bevorzugt zwischen Gehalten von 100-10.000 Einheiten Phytase/kg Futtermittel und weniger als 0,225% anorganischem Phosphor, bevorzugter Gehalte von 150-10.000 Einheiten Phytase/kg Futtermittel und weniger als 0,15% anorganischem Phosphor, noch bevorzugter Gehalte von 200-20.000 Einheiten Phytase/kg Futtermittel und keinen zusätzlichen Zusatz an anorganischem Phosphor, betragen.Phytase formulations according to the invention can also be combined with other ingredients, thereby creating new and particularly advantageous feed compositions. As stated above, since the availability of vegetable phosphate in soybean meal and cereals as a result of binding to phytic acid is low, inorganic phosphate is added to the feed to ensure adequate phosphorus supply to the animals. However, these feeds contain too much total phosphate, leading to pollution of the environment with phosphate. Specifically, the animal feed of the present invention comprises the combination of a phytase of the invention with animal feed ingredients to yield a feed containing substantially reduced levels of added inorganic phosphorus. In a preferred embodiment, the feed according to the invention comprises typical feed ingredients, micronutrients, trace elements, vitamins, etc. as well as an effective amount of phytase and inorganic phosphorus, the amounts of phytase and phosphorus being between 50 and 20,000 units Phytase / kg feed and less than 0.45% inorganic phosphorus, preferably between levels of 100-10,000 units phytase / kg feed and less than 0,225% inorganic phosphorus, more preferred levels of 150-10,000 units phytase / kg feed and less than 0, 15% inorganic phosphorus, more preferably levels of 200-20,000 units phytase / kg feed and no additional addition of inorganic phosphorus.
Die Erfindung betrifft auch Verfahren zur Verbesserung der Gewichtszunahme und des Futterverwertungsvermögens (Feed Conversion Ratio, FCR) in der Tierernährung sowie die Verwendung der erfindungsgemäßen Phytasen in diesen Verfahren. Eine erfindungsgemäße Phytase erlaubt verbesserte Gewichtszunahmen und ein verbessertes Futterverwertungsvermögen, insbesondere im Zusammenhang mit Futter, das wenig anorganisches Phosphat enthält. Gemäß den erfindungsgemäßen Verfahren kann der Gehalt an anorganischem Phosphat in Futter unter Gehalte von 0,45%, bevorzugt unter 0,225%, gesenkt werden. Bevorzugt wird kein anorganisches Phosphat zugesetzt. Durch eine erhöhte Phosphat-Verfügbarkeit in Folge des Zusatzes des erfindungsgemäßen Enzyms kann die Knochenmineralisation der Tiere signifikant verbessert werden, was insbesondere bei rasch wachsenden Tieren von großer Bedeutung ist.The invention also relates to methods for improving weight gain and feed conversion ratio (FCR) in animal nutrition and to the use of the phytases according to the invention in these methods. A phytase of the invention allows for improved weight gain and improved feed utilization, especially in the context of feed containing little inorganic phosphate. According to the methods of the invention, the content of inorganic phosphate in feed can be reduced below levels of 0.45%, preferably below 0.225%. Preferably, no inorganic phosphate is added. Increased availability of phosphate as a result of the addition of the enzyme of the invention, the bone mineralization of the animals can be significantly improved, which is particularly important in fast-growing animals of great importance.
Gemäß einer noch weiteren Ausführungsform betrifft die Erfindung die Verwendung des erfindungsgemäßen Enzyms beim Backen, wodurch Entwicklung, Elastizität und/oder Stabilität des Teigs und/oder das Volumen, die Krumenstruktur und/oder der Widerstand des Backguts gegenüber dem Altbackenwerden verbessert wird. Obwohl das erfindungsgemäße Enzympräparat zur Herstellung von Teig oder gebackenen Produkten aus beliebigen Typen von Mehl, z. B. auf der Basis von Roggen, Gerste, Hafer oder Mais verwendet werden kann, erwies sich das erfindungsgemäße Enzympräparat als besonders nützlich bei der Herstellung von Teigen oder Backprodukten aus Weizen oder aus einem wesentlichen Weizenanteil. Die Backprodukte, die mit einem erfindungsgemäßen Enzympräparat hergestellt werden können, umfassen Brot, Brötchen, Baguette und dergleichen. Zum Backen kann das erfindungsgemäße Enzympräparat mit einer weiteren Enzymaktivität wie z. B. Xylanase, Lipase, Amylase, Oxidase oder Laccase neben der Phytase verwendet werden oder kann in Kombinationen mit weiteren Enzymen wie Lipase, Amylase, Oxidase (z. B. Glucoseoxidase, Peroxidase), verwendet werden.According to yet another embodiment, the invention relates to the use of the enzyme according to the invention in baking, whereby the development, elasticity and / or stability of the dough and / or the volume, the crumb structure and / or the resistance of the baked product to staling is improved. Although the enzyme preparation according to the invention for the preparation of dough or baked products from any type of flour, z. B. based on rye, barley, oats or corn can be used, the enzyme preparation according to the invention proved to be particularly useful in the preparation of doughs or bakery products from wheat or from a substantial proportion of wheat. The bakery products that can be made with an enzyme preparation of the invention include bread, rolls, Baguette and the like. For baking, the enzyme preparation of the invention with a further enzyme activity such as. Xylanase, lipase, amylase, oxidase or laccase besides the phytase, or may be used in combination with other enzymes such as lipase, amylase, oxidase (e.g., glucose oxidase, peroxidase).
Des weiteren betrifft die Anmeldung folgende Gegenstände:
- 1. Rekombinantes DNA-Molekül, das nach Expression in einer prokaryotischen oder eukaryotischen Wirtszelle ein Polypeptid mit Phytaseaktivität codiert, wobei das rekombinante DNA-Molekül eine DNA-Sequenz, ausgewählt aus
- a) DNA-Sequenzen, die ein Polypeptid mit Phytaseaktivität codieren, das durch Variation der reifen Wildtyp-E. coli-Phytasesequenz erhalten worden ist, wobei die Variation ausgewählt ist aus
- i) der Mutation Lysin → Asparaginsäure an Position 74 (K74D), und/oder
- ii) einer Kombination der Mutationen Asparagin → Arginin an Position 139 (N139R) und Asparaginsäure → Glutaminsäure an Position 142 (D142E), und/oder
- iii) einer Kombination der Mutationen Leucin → Isoleucin an Position 145 (L1451) und Leucin → Isoleucin an Position 198 (L1981), und/oder
- iv) einer Mutation Valin → Prolin an Position 200 (V200P), und/oder
- v) einer N- oder C-terminalen oder einer N- und C-terminalen Anfügung eines Sequenzabschnitts der sauren Phosphatase von Aspergillus niger var. awamori oder der Phytase von Aspergillus niger,
- b) DNA-Sequenzen, die eine Homologie von 70% bis 100% zu den Sequenzen gemäß a) aufweisen,
- c) DNA-Sequenzen, die aufgrund der Degeneriertheit des genetischen Codes mit den Sequenzen gemäß a) und b) verwandt sind,
- a) DNA-Sequenzen, die ein Polypeptid mit Phytaseaktivität codieren, das durch Variation der reifen Wildtyp-E. coli-Phytasesequenz erhalten worden ist, wobei die Variation ausgewählt ist aus
- 2. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die DNA-Sequenz die Variation N139R und D142E des codierten Polypeptids umfasst. - 3. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die DNA-Sequenz mindestens die Variation V200P des codierten Polypeptids umfasst. - 4. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die DNA-Sequenz die Variation K74D des codierten Polypeptids umfasst. - 5. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die DNA-Sequenz mindestens die Variation L1451 und L198I des codierten Polypeptids umfasst. - 6. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die DNA-Sequenz die Variation K74D, N139R, D142E, V200P des codierten Polypeptids umfasst. - 7. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die N-terminale Anfügung des codierten Polypeptids die Sequenz FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS umfasst. - 8. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die C-terminale Anfügung des codierten Polypeptids die Sequenz LSFWWNYNTT TELNYRSSPI ACQEGDAMD umfasst. - 9. Rekombinantes DNA-
Molekül nach Gegenstand 1, dadurch gekennzeichnet, dass die N-terminale Anfügung des codierten Polypeptids die Sequenz FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS und die C-terminale Anfügung des codierten Polypeptids die Sequenz LSFWWNYNTT TELNYRSSPI ACQEGDAMD umfasst. - 10. Rekombinantes DNA-Molekül nach einem der Gegenstände 1 bis 8, dadurch gekennzeichnet, dass es eine Sequenz ausgewählt aus den Sequenzen SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 umfasst.
- 11. Polypeptid, das Phytaseaktivität besitzt und von einem rekombinanten DNA-Molekül nach einem der Gegenstände 1 bis 10 codiert wird oder durch Expression einer damit transformierten Wirtszelle erhalten wird.
- 12. Polypeptid nach Gegenstand 11, dadurch gekennzeichnet, dass es eine Sequenz, ausgewählt aus SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 umfasst.
- 13. DNA-Konstrukt mit der Fähigkeit, nach dem Einführen in eine geeignete Wirtszelle die Expression eines mutierten Phytasegens in einem Wirt zu steuern, dadurch gekennzeichnet, dass es gegebenenfalls einen Promotor, gegebenenfalls Signal- und Markersequenzen, eine DNA-Sequenz nach einem der Ansprüche 1 bis 10, einen Terminator und gegebenenfalls 5'- und 3'-flankierende Sequenzen enthält.
- 14. DNA-Konstrukt nach Gegenstand 13, dadurch gekennzeichnet, dass es sich bei dem Promotor um den Cellobiohydrolase-I-, den Cellobiohydrolase-II-, den Amylase-, den Glucoamylase-, den Xylanase- oder den Enolase-Promotor handelt.
- 15. DNA-Konstrukt nach Gegenstand 14, dadurch gekennzeichnet, dass es sich bei der Signalsequenz und/oder den 5'- und 3'-flankierenden Sequenzen um gegebenenfalls modifizierte Phytase-Signalsequenzen aus Aspergillus niger handelt.
- 16. Vektor mit der Fähigkeit, eine Wirtszelle zu transformieren, dadurch gekennzeichnet, dass der Vektor ein Konstrukt nach Gegenstand 13 enthält.
- 17. Vektor nach Gegenstand 16, dadurch gekennzeichnet, dass er ausgewählt ist aus dem Plasmid pET-PhyM2, hinterlegt unter der Hinterlegungsnummer DSM 18715, dem Plasmid pUC-PhyM3, hinterlegt unter der Hinterlegungsnummer DSM 18717, dem Plasmid pET-PhyM7, hinterlegt unter der Hinterlegungsnummer DSM 18716, dem Plasmid pUC-PhyM9, hinterlegt unter der Hinterlegungsnummer DSM 18718, dem Plasmid pUC-PhyM10, hinterlegt unter der Hinterlegungsnummer DSM 18719, dem Plasmid pPhy2005, hinterlegt unter der Hinterlegungsnummer DSM 18720, dem Plasmid pPhy2006, hinterlegt unter der Hinterlegungsnummer DSM 18721.
- 18. Transformierte Wirtszelle, ausgewählt aus Pilz, Hefe, Bakterien und Säugerzellen, enthaltend ein rekombinantes DNA-Molekül nach einem der Gegenstände 1 bis 10 und ausgestattet mit der Fähigkeit, ein Polypeptid mit Phytaseaktivität zu exprimieren.
- 19. Transformierte Wirtszelle nach Gegenstand 18, dadurch gekennzeichnet, dass sie der Gattung Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor oder Penicillium angehört.
- 20. Verfahren zur Produktion von Phytase, dadurch gekennzeichnet, dass eine transformierte Wirtszelle nach Gegenstand 18 oder 19 unter Bedingungen gezüchtet wird, die der Bildung von Phytase förderlich sind und die so produzierte Phytase isoliert wird.
- 21. Zusammensetzung, umfassend ein Polypeptid nach Gegenstand 11 oder 12, gegebenenfalls zusammen mit weiteren Hilfs- und/oder Wirkstoffen.
- 22. Zusammensetzung nach Gegenstand 21, dadurch gekennzeichnet, dass sie Magermilchpulver und/oder Calciumpropionat enthält.
- 23. Zusammensetzung nach Gegenstand 21, dadurch gekennzeichnet, dass die Zusammensetzung eine Nahrungsmittel- oder Futtermittelzusammensetzung ist.
- 24. Zusammensetzung nach Gegenstand 21, 22 oder 23, dadurch g e - kennzeichnet, dass die Zusammensetzung ein Backmittel ist.
- 25. Verwendung eines Polypeptids nach Gegenstand 11 oder 12 zur Herstellung eines Präparats zur Verbesserung der Phosphatverwertung aus der Nahrung bei Tieren und Menschen.
- 26. Verwendung eines Polypeptids nach Gegenstand 11 oder 12 zur Verbesserung der rheologischen Eigenschaften von Teigen zur Herstellung von Backwaren.
- 27. Verwendung einer Variation der reifen Wildtyp-E. coli-Phytasesequenz, ausgewählt aus
- i) der Mutation Lysin → Asparaginsäure an Position 74 (K74D), und/oder
- ii) einer Kombination der Mutationen Asparagin → Arginin an Position 139 (N139R) und Asparaginsäure → Glutaminsäure an Position 142 (D142E), und/oder
- iii) einer Kombination der Mutationen Leucin → Isoleucin an Position 145 (L1451) und Leucin → Isoleucin an Position 198 (L1981), und/oder
- iv) einer Mutation Valin → Prolin an Position 200 (V200P), und/oder
- v) einer N- oder C-terminalen oder einer N- und C-terminalen Anfügung eines Sequenzabschnitts der sauren Phosphatase von Aspergillus niger var. awamori oder der Phytase von Aspergillus niger
- 28. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation K74D ist.
- 29. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation N139R und D142E ist.
- 30. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation L145I und L198I ist.
- 31. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation V200P ist.
- 32. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation K74D, N139R, D142E, V200P ist
- 33. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation die N-terminale Anfügung der Sequenz FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS ist.
- 34. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation die C-terminale Anfügung die Sequenz LSFWWNYNTT TELNYRSSPI ACQEGDAMD ist.
- 35. Verwendung nach Gegenstand 27, dadurch gekennzeichnet, dass die Variation die N-terminale Anfügung der Sequenz FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS und die C-terminale Anfügung die Sequenz LSFWWNYNTT TELNYRSSPI ACQEGDAMD ist.
- A recombinant DNA molecule which, when expressed in a prokaryotic or eukaryotic host cell, encodes a polypeptide having phytase activity, wherein the recombinant DNA molecule comprises a DNA sequence selected from
- a) DNA sequences encoding a polypeptide with phytase activity obtained by variation of the wild-type mature E. coli phytase sequence, the variation being selected from
- i) the mutation lysine → aspartic acid at position 74 (K74D), and / or
- ii) a combination of the mutations asparagine → arginine at position 139 (N139R) and aspartic acid → glutamic acid at position 142 (D142E), and / or
- iii) a combination of the mutations leucine → isoleucine at position 145 (L1451) and leucine → isoleucine at position 198 (L1981), and / or
- iv) a mutation valine → proline at position 200 (V200P), and / or
- v) an N- or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. awamori or the phytase of Aspergillus niger,
- b) DNA sequences having a homology of 70% to 100% to the sequences according to a),
- c) DNA sequences related to the sequences according to a) and b) due to the degeneracy of the genetic code,
- a) DNA sequences encoding a polypeptide with phytase activity obtained by variation of the wild-type mature E. coli phytase sequence, the variation being selected from
- 2. Recombinant DNA molecule according to
item 1, characterized in that the DNA sequence comprises the variation N139R and D142E of the encoded polypeptide. - 3. Recombinant DNA molecule according to
item 1, characterized in that the DNA sequence comprises at least the variation V200P of the encoded polypeptide. - 4. Recombinant DNA molecule according to
item 1, characterized in that the DNA sequence comprises the variation K74D of the encoded polypeptide. - 5. Recombinant DNA molecule according to
item 1, characterized in that the DNA sequence comprises at least the variation L1451 and L198I of the encoded polypeptide. - 6. Recombinant DNA molecule according to
item 1, characterized in that the DNA sequence comprises the variation K74D, N139R, D142E, V200P of the encoded polypeptide. - 7. Recombinant DNA molecule according to
item 1, characterized in that the N-terminal addition of the encoded polypeptide the sequence FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS comprises. - 8. Recombinant DNA molecule according to
item 1, characterized in that the C-terminal addition of the encoded polypeptide comprises the sequence LSFWWNYNTT TELNYRSSPI ACQEGDAMD. - 9. Recombinant DNA molecule according to
item 1, characterized in that the N-terminal addition of the encoded polypeptide comprises the sequence FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS and the C-terminal addition of the encoded polypeptide comprises the sequence LSFWWNYNTT TELNYRSSPI ACQEGDAMD. - 10. Recombinant DNA molecule according to any one of
items 1 to 8, characterized in that it comprises a sequence selected from the sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21. - A polypeptide having phytase activity which is encoded by a recombinant DNA molecule of any one of
items 1 to 10 or obtained by expression of a host cell transformed therewith. - 12. Polypeptide according to item 11, characterized in that it has a sequence selected from SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO : 20, SEQ ID NO: 22.
- A DNA construct having the ability to direct expression of a mutated phytase gene in a host upon introduction into a suitable host cell, characterized in that it optionally comprises a promoter, optionally signal and marker sequences, a DNA sequence according to any one of
claims 1 to 10, a terminator and optionally 5 'and 3' flanking sequences. - 14. DNA construct according to item 13, characterized in that the promoter is the cellobiohydrolase I, the cellobiohydrolase II, the amylase, the glucoamylase, the xylanase or the enolase promoter.
- 15. DNA construct according to item 14, characterized in that the signal sequence and / or the 5 'and 3' flanking sequences are optionally modified phytase signal sequences from Aspergillus niger .
- 16. Vector having the ability to transform a host cell, characterized in that the vector contains a construct according to item 13.
- 17. Vector according to item 16, characterized in that it is selected from the plasmid pET-PhyM2, deposited under the accession number DSM 18715, the plasmid pUC-PhyM3, deposited under the accession number DSM 18717, the plasmid pET-PhyM7, deposited under the Deposit number DSM 18716, the plasmid pUC-PhyM9, deposited under the accession number DSM 18718, the plasmid pUC-PhyM10, deposited under the accession number DSM 18719, the plasmid pPhy2005, deposited under the accession number DSM 18720, the plasmid pPhy2006, deposited under the accession number DSM 18,721th
- 18. Transformed host cell selected from fungus, yeast, bacteria and mammalian cells containing a recombinant DNA molecule according to any one of
items 1 to 10 and provided with the ability to express a polypeptide having phytase activity. - 19. Transformed host cell according to item 18, characterized in that it belongs to the genus Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor or Penicillium.
- 20. A process for the production of phytase, characterized in that a transformed host cell according to item 18 or 19 is grown under conditions conducive to the formation of phytase and the phytase thus produced is isolated.
- 21. A composition comprising a polypeptide according to item 11 or 12, optionally together with other excipients and / or active substances.
- 22. Composition according to Article 21, characterized in that it contains skimmed milk powder and / or calcium propionate.
- Composition according to item 21, characterized in that the composition is a food or feed composition.
- 24. Composition according to Article 21, 22 or 23, characterized in that the composition is a baking agent.
- 25. Use of a polypeptide according to item 11 or 12 for the preparation of a preparation for improving the phosphate utilization from food in animals and humans.
- 26. Use of a polypeptide according to item 11 or 12 for improving the rheological properties of doughs for the production of baked goods.
- 27. Use of a variation of the wild-type mature E. coli phytase sequence selected from
- i) the mutation lysine → aspartic acid at position 74 (K74D), and / or
- ii) a combination of the mutations asparagine → arginine at position 139 (N139R) and aspartic acid → glutamic acid at position 142 (D142E), and / or
- iii) a combination of the mutations leucine → isoleucine at position 145 (L1451) and leucine → isoleucine at position 198 (L1981), and / or
- iv) a mutation valine → proline at position 200 (V200P), and / or
- v) an N- or C-terminal or an N- and C-terminal addition of a sequence portion of the acid phosphatase of Aspergillus niger var. awamori or the phytase of Aspergillus niger
- 28. Use according to item 27, characterized in that the variation is K74D.
- Use according to item 27, characterized in that the variation is N139R and D142E.
- Use according to item 27, characterized in that the variation is L145I and L198I.
- Use according to item 27, characterized in that the variation is V200P.
- Use according to item 27, characterized in that the variation is K74D, N139R, D142E, V200P
- 33. Use according to item 27, characterized in that the variation is the N-terminal addition of the sequence FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS.
- Use according to item 27, characterized in that the variation is the C-terminal addition the sequence LSFWWNYNTT TELNYRSSPI ACQEGDAMD.
- 35. Use according to item 27, characterized in that the variation is the N-terminal addition of the sequence FSYGAAIPQS TQEKQFSQEF RDGYSILKHY GGNGPYSERV SYGIARDPPTS and the C-terminal addition is the sequence LSFWWNYNTT TELNYRSSPI ACQEGDAMD.
Die beigefügten Figuren erläutern die Erfindung näher:
- Fig. 1:
- Plasmidkarte von pET-PhyM2
- Fig. 2:
- Plasmidkarte von pAB490-PhyM2
- Fig. 3:
- Plasmidkarte von pUC-PhyM3
- Fig. 4:
- Plasmidkarte von pAB489-PhyM3
- Fig. 5:
- Plasmidkarte von pUC-PhyM10
- Fig. 6:
- Plasmidkarte von pAB600-PhyM10
- Fig. 7:
- Plasmidkarte von pPhy2005
- Fig. 8:
- Plasmidkarte von pAB-Phy2005
- Fig. 9:
- Plasmidkarte von pPhy2006
- Fig. 10:
- Plasmidkarte von pAB-Phy2006
- Fig. 11:
- Sequenzvergleich einzelner erfindungsgemäßer Mutanten und Varianten auf Basis der Wild-Typ Sequenz (Dassa). Die Mutationen bzw. Variationen sind hervorgehoben.
- Fig. 1:
- Plasmid map of pET-PhyM2
- Fig. 2:
- Plasmid map of pAB490-PhyM2
- 3:
- Plasmid map of pUC-PhyM3
- 4:
- Plasmid map of pAB489-PhyM3
- Fig. 5:
- Plasmid map of pUC-PhyM10
- Fig. 6:
- Plasmid map of pAB600-PhyM10
- Fig. 7:
- Plasmid card from pPhy2005
- Fig. 8:
- Plasmid map of pAB-Phy2005
- Fig. 9:
- Plasmid card from pPhy2006
- Fig. 10:
- Plasmid map of pAB-Phy2006
- Fig. 11:
- Sequence comparison of individual inventive mutants and variants based on the wild-type sequence (Dassa). The mutations or variations are highlighted.
Die nachstehenden Beispiele erläutern die Erfindung näher.The following examples illustrate the invention in more detail.
Die Phytaseaktivität wurde in einem Assay-Gemisch aus 0,5% Phytinsäure (etwa 5 mM), 200 mM Natriumcitrat, pH 5,0, gemessen. Nach 15-minütiger Inkubation bei 37°C wurde die Reaktion durch Zugabe eines gleichen Volumens 15% Trichloressigsäure gestoppt. Die freigesetzten Phosphationen wurden durch Mischen von 100 µl des Assay-Gemisches mit 900 µl H2O und 1 ml 0,6 M H2SO4, 2% Ascorbinsäure und 0,5% Ammoniummolybdat nach Inkubation bei 50°C und einer Dauer von 20 min, bei 820 nm quantitativ bestimmt. Kaliumphosphat-Standardlösungen wurden als Referenz verwendet.Phytase activity was measured in an assay mixture of 0.5% phytic acid (about 5 mM), 200 mM sodium citrate, pH 5.0. After 15 minutes incubation at 37 ° C, the reaction was stopped by adding an equal volume of 15% trichloroacetic acid. The liberated phosphate ions were prepared by mixing 100 μl of the assay mixture with 900 μl of H 2 O and 1 ml of 0.6 MH 2 SO 4 , 2% ascorbic acid and 0.5% ammonium molybdate after incubation at 50 ° C. and a duration of 20 min, quantified at 820 nm. Potassium phosphate standard solutions were used as reference.
Die Konstruktion des Plasmids pAB490-PhyM2 erfolgte durch folgende Schritte:The construction of the plasmid pAB490-PhyM2 was carried out by the following steps:
Das Plasmid pET-PhyM2 enthält die E. coli Phytase Sequenz (Dassa et al. 1990, Accession Nr. M58740) mit den Änderungen der Aminosäuren N139R und D142E.The plasmid pET-PhyM2 contains the E. coli phytase sequence (Dassa et al., 1990, Accession No. M58740) with the changes of amino acids N139R and D142E.
Die DNA Sequenz, die mit dem CAG (Gln)-Codon in Position 1 ein offenes Leseraster von 1230 bp umfasst und ein Enzym mit 410 Aminosäuren codiert, wurde unter Verwendung des Codon-Gebrauchs von T. reesei (http://www.kazusa.or.jp/codon) in das Plasmid pET26b(+) (Novagen, Deutschland, modifiziert durch Brain, Deutschland) eingefügt. Zur Realisierung der Mutagenesen wurde eine PCR-basierte Oligonucleotid-gerichtete Methode angewandt. Ausgehend von dem Plasmid, das das Gen kodierend für die Wildtyp-Dassa-Aminosäuresequenz enthält, wurden für jedes auszutauschende Triplet zwei komplementäre Primer mit den jeweiligen Mutationen synthetisiert, wobei die Mutation jeweils in der Mitte der Primer lokalisiert ist. Mithilfe der beiden Primer wurde dann das gesamte Plasmid amplifiziert. Das erhaltene Plasmid mit den Mutationen für den Genotyp M2 hat die Bezeichnung pET-PhyM2.The DNA sequence comprising an open reading frame of 1230 bp with the CAG (Gln) codon at
Zur Konstruktion wurden die folgenden Primer benutzt.
a) Für die Mutationen an Position 139:
a) For the mutations at position 139:
Das Plasmid ist in
Zur Konstruktion des Plasmids pAB490-PhyM2 wurde das für E. coli-Phytase codierende Gen PhyM2 aus dem Plasmid pET-PhyM2 mittels PCR amplifiziert. Das PCR-Produkt wurde mit den Restriktionsenzymen SpeI und PacI geschnitten und in die Spel- und Pacl-Schnittstellen nach dem T. reesei cbhll-Genfragment in dem Plasmid pAB490 inseriert. Dadurch entstand ein offenes Leseraster, das die Fusion CBHII-Kexll-PhyM2 codiert.For the construction of the plasmid pAB490-PhyM2, the PhyM2 gene coding for E. coli phytase was amplified from the plasmid pET-PhyM2 by PCR. The PCR product was cut with the restriction enzymes SpeI and PacI and inserted into the Spel and PacI sites after the T. reesei cbhlI gene fragment in plasmid pAB490. This gave rise to an open reading frame encoding the CBHII-KexII-PhyM2 fusion.
Das entstandene Plasmid hat die Bezeichnung pAB490-PhyM2 (
Das Plasmid pAB490 basiert auf pUC18 und enthält das cbhll Genfragment (
Die aus dem Plasmid pAB490-PhyM2 isolierte Expressionskassette enthält das folgende genetische Material:
- cbhl (Cellobiohydrolase I) Promotor: Das 2,2 kb EcoRI/SacII-Fragment, das den cbhI-Promotor enthält, stammt von Trichoderma reesei QM6a. Der PromotorBereich funktioniert auch als homologe DNA (zusammen mit dem cbhl 3'-Fragment; siehe nachstehend), um die Integration der transformierenden DNA in den cbhl Lokus zu steuern.
- cbhII-Genfragment: Das 307-bp cbhll Genfragment mit seiner Signalsequenz steht unmittelbar unter der Kontrolle des cbhI-Promotors.
- cbhl (cellobiohydrolase I) promoter: The 2.2 kb EcoRI / SacII fragment containing the cbhI promoter is from Trichoderma reesei QM6a. The promoter region also functions as a homologous DNA (along with the cbhl 3 'fragment, see below) to direct integration of the transforming DNA into the cbhl locus.
- cbhII gene fragment: The 307-bp cbhll gene fragment with its signal sequence is immediately under the control of the cbhI promoter.
Die E. coli-Phytase Sequenz, die die Mutation der Aminosäuren N139R und D142E enthält, ist mittels einer Kexll-Schnittstelle am 3'-Ende des cbhll-Genfragments fusioniert. Die Kex-Sequenz enthält folgende Aminosäuren: RTLVKR.The E. coli phytase sequence containing the mutation of amino acids N139R and D142E is fused at the 3 'end of the cbhll gene fragment via a KexII site. The Kex sequence contains the following amino acids: RTLVKR.
cbhl Terminator: Das 0,75 kb lange BamHI/Stul-Fragment, das den cbhl-Terminator enthält, wurde im Anschluss an die E. coli-Phytase hinzugefügt, um die Termination der Transkription zu gewährleisten.cbhl Terminator: The 0.75 kb BamHI / Stul fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
amdS-Gen: Das Gen inkl. seines Promotors und seines Terminators wurde aus Aspergillus nidulans VH1-TRSX6 isoliert und codiert für Acetamidase (
cbhl 3' Fragment: Das Fragment (1,7 kb, BamHI/EcoRI, beginnend 1,4 kb nach dem Stopcodon des Gens) wurde aus T. reesei ALKO2466 isoliert. Der Stamm ALKO2466 stammt von dem Stamm ALKO233 (
Die Sequenz des Plasmids pAB490-PhyM2 wurde durch Kartierung mittels Restriktionsenzymen und Sequenzierung bestätigt.The sequence of plasmid pAB490-PhyM2 was confirmed by restriction enzyme mapping and sequencing.
Das Plasmid pET-PhyM7 enthält die E. coli-Phytase (Dassa et al. 1990, Accession Nr. M58740) Sequenz unter Verwendung des Codon-Gebrauchs von T. reesei (http://www.kazusa.or.jp/codon) mit der Mutation K74D. Die Konstruktion sowie die Klonierung des Plasmids pET-PhyM7 erfolgte analog zu der in Beispiel 2 beschriebenen Herstellung des Plasmids pET-PhyM2. Die zur Einfügung der Mutationen an Aminosäureposition 74 benutzten Primer sind:
Das Plasmid pET-PhyM7_ist unter der Hinterlegungsnummer DSM 18716 am 18.10.2006 hinterlegt worden.The plasmid pET-PhyM7_ has been deposited under the accession number DSM 18716 on 18.10.2006.
Die Konstruktion sowie die Klonierung des Plasmids pAB490-PhyM7 erfolgte analog zu der in Beispiel 2 beschriebenen Herstellung des Plasmids pAB490-PhyM2. Die Sequenz des Plasmids pAB490-PhyM7 wurde durch Sequenzierung bestätigt. Die aus dem Plasmid pAB490-PhyM7 isolierte Expressionskassette enthält daher mit Ausnahme der neuen spezifischen Phytasesequenz des Genotyps PhyM7 die gleichen Elemente wie in Beispiel 2 beschrieben.The construction and the cloning of the plasmid pAB490-PhyM7 was carried out analogously to the preparation of the plasmid pAB490-PhyM2 described in Example 2. The sequence of the plasmid pAB490-PhyM7 was determined by sequencing approved. The expression cassette isolated from the plasmid pAB490-PhyM7 therefore contains the same elements as described in Example 2, with the exception of the new specific phytase sequence of the genotype PhyM7.
Die Phytasevariante PhyM3 enthält die E. coli Phytase (Dassa et al. 1990, Accession Nr. M58740) Sequenz unter Verwendung des Codon-Gebrauchs von T. reesei (http://www.kazusa.or.jp/codon) mit der Mutation V200P. Die DNA-Sequenz mit dem CAG (Gln)-Codon in Position 1 umfasst ein offenes Leseraster von 1230 bp und codiert ein Enzym mit 410 Aminosäuren. Das 18 Aminosäuren lange Signalpeptid der Phytase von A. niger wurde verwendet, um die Phytasemutante von E. coli aus Trichoderma reesei zu sezernieren.The phytase variant PhyM3 contains the E. coli phytase (Dassa et al., 1990, Accession No. M58740) sequence using the codon usage of T. reesei (http://www.kazusa.or.jp/codon) with the mutation V200P. The DNA sequence with the CAG (Gln) codon in
Ausgehend von dem Plasmid, das das Gen kodierend für die Wildtyp-Dassa-Aminosäuresequenz enthält, wurde die Mutation V200P mittels der PCR Methode analog dem Prinzip von
Die Konstruktion pAB489-PhyM3 (
Die Konstruktion des Plasmids pAB489 erfolgte durch folgende Schritte:
- Aus dem Plasmid pALK487 (
WO94/28117 WO 93/24621
- From the plasmid pALK487 (
WO94 / 28117 WO 93/24621
Die aus dem pAB489-PhyM3 isolierte Expressionskassette enthält das folgende genetische Material:
- Cbhl (Cellobiohydrolase I)-Promotor: Das 2,2 kb EcoRI/SacII-Fragment, das den cbhI-Promotor enthält, stammt von Trichoderma reesei QM6a. Der Promotor-Bereich funktioniert auch als homologe DNA (zusammen mit dem cbhl-3'-Fragment; siehe nachstehend), um die Integration der transformierenden DNA in den cbhl-Lokus zu steuern.
- Cbhl (cellobiohydrolase I) promoter: The 2.2 kb EcoRI / SacII fragment containing the cbhI promoter is from Trichoderma reesei QM6a. The promoter region also functions as a homologous DNA (along with the cbhl-3 'fragment, see below) to direct integration of the transforming DNA into the cbh1 locus.
Signalsequenz: Das modifizierte Signalpeptid der A. niger-Phytase wurde verwendet, um die E. coli-Phytase aus Trichoderma reesei zu sezernieren.Signal sequence: The modified signal peptide of A. niger- phytase was used to secrete the E. coli phytase from Trichoderma reesei .
Die E. coli-Phytase mit der Mutation V200P Sequenz inkl. der A. niger-Phytase-Signalsequenz wurde zwischen den cbhl Promotor und den cbhl Terminator inseriert.The E. coli phytase with the mutation V200P sequence including the A. niger phytase signal sequence was inserted between the cbhl promoter and the cbhl terminator.
cbhI-Terminator: Das 0,75 kb lange BamHI/StuI-Fragment, das den cbhl-Terminator enthält, wurde im Anschluss an die E. coli-Phytase hinzugefügt, um die Termination der Transkription zu gewährleisten.cbhI terminator: The 0.75 kb BamHI / StuI fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
amdS-Gen: Das Gen inkl. seines Promotors und seines Terminators wurde aus Aspergillus nidulans VH1-TRSX6 isoliert und codiert für Acetamidase (
cbhl 3' Fragment: Das Fragment (1,7 kb, BamHI/EcoRI, beginnend 1,4 kb nach dem Stopcodon des Gens) wurde aus T. reesei ALKO2466 isoliert. Der Stamm ALKO2466 stammt von dem Stamm ALKO233 (
Das Phytasevariante PhyM3 Gen wurde in das Plasmid pAB2004 kloniert. Das erhaltene Plasmid hat die Bezeichnung pUC-PhyM3 (
Das Plasmid pAB2004 basiert auf dem Plasmid pUC18 und wurde durch Einfügen weitere Schnittstellen in die HindIII-EcoRI sites hergestellt.The plasmid pAB2004 is based on the plasmid pUC18 and was prepared by inserting additional sites into the HindIII-EcoRI sites.
Die Phytasevariante PhyM9 enthält die E. coli Phytase (Dassa et al. 1990, Accession Nr. M58740) Sequenz als synthetisches Gen unter Verwendung des Codon-Gebrauchs von T. reesei (http://www.kazusa.or.jp/codon) mit den Mutationen L1451 und L1981. Die DNA-Sequenz mit dem CAG (Gln)-Codon in Position 1 umfasst ein offenes Leseraster von 1230 bp und codiert ein Enzym mit 410 Aminosäuren.The phytase variant PhyM9 contains the E. coli phytase (Dassa et al., 1990, Accession No. M58740) Sequence as a synthetic gene using the codon usage of T. reesei (http://www.kazusa.or.jp/codon) with mutations L1451 and L1981. The DNA sequence with the CAG (Gln) codon in
In dem Plasmid pAB489-PhyM9 steht das Gen kodierend für die E. coli Phytasevariante PhyM9 mit der A. niger Phytase Signalsequenz (MGVSAVLLPLYLLSGVTS)
Die Konstruktion von pAB489-PhyM9 und pUC-PhyM9 erfolgte analog zu Plasmid pAB489-PhyM3 und pUC-PhyM3 in Beispiel 4 und enthält damit mit Ausnahme der A. niger Signalsequenz und der spezifischen E. coli Phytasesequenz die gleichen genetischen Elemente.The construction of pAB489-PhyM9 and pUC-PhyM9 was analogous to plasmid pAB489-PhyM3 and pUC-PhyM3 in Example 4 and thus contains, with the exception of the A. niger signal sequence and the specific E. coli phytase sequence the same genetic elements.
Das Plasmid pUC-PhyM9 ist unter der Hinterlegungsnummer DSM 18718 am 18.10.2006 hinterlegt worden.The plasmid pUC-PhyM9 has been deposited under the accession number DSM 18718 on 18.10.2006.
Das Plasmid pAB600-PhyM10 enthält die E. coli Phytase Sequenz mit den Änderungen der Aminosäuren K74D, N139R, D142E und V200P. Aus den Plasmiden pAB490-PhyM2, pAB490-PhyM7 und pAB489-PhyM3 wurde das Gen kodierend für die Phytasevariante PhyM10 mittels der PCR Methode analog dem Prinzip von
Das entstandene Plasmid pAB600-PhyM10 (
In dem Plasmid pAB600-PhyM10 wurde die E. coli Phytase Sequenz mittels einer KexII-Schnittstelle am 3'Ende des CBHII-Genfragments fusioniert. Die Kex Sequenz enthält folgende Aminosäuren RTLVKR.In the plasmid pAB600-PhyM10, the E. coli phytase sequence is fused at the 3 'end of the CBHII gene fragment via a KexII site. The Kex sequence contains the following amino acids RTLVKR.
Die Konstruktion des Plasmids pAB600 erfolgte durch folgende Schritte:
- Das Plasmid pAB1280 basiert auf pUC18 und enthält das cbhII-Genfragment (
Nucleotide 1-307 entspricht Aminosäure M1 bis S86, Teeri et al, 1987, Gene 51: 43-52 WO 94/28117
- The plasmid pAB1280 is based on pUC18 and contains the cbhII gene fragment (
Nucleotide 1-307 corresponds to amino acid M1 to S86, Teeri et al, 1987, Gene 51: 43-52 WO 94/28117
Aus dem Plasmid p3SR2 (
Für die Amplifizierung des amdS-Gens wurden folgende Primer aus der Sequenzinformation des A. nidulans-Acetamidase-Gens abgeleitet:
Das erhaltene Plasmid hat die Bezeichnung pAB600 wurde durch Kartierung und Sequenzierung bestätigt.The resulting plasmid has been designated pAB600 has been confirmed by mapping and sequencing.
Das Gen der Phytasevariante PhyM10 wurde in das Plasmid pAB2004 kloniert. Das Plasmid pUC-PhyM10 ist in
Die Konstruktion des Plasmids pAB-Phy2005 erfolgte durch folgende Schritte:The construction of the plasmid pAB-Phy2005 was carried out by the following steps:
Das Plasmid pPhy2005 enthält die Fusion aus dem A. niger var. awamorisaure-Phosphatase (ap)-Gen (
Die Fusion aus der A. niger var. awamori sauren Phosphatase Sequenz und der unter Verwendung des Codon-Gebrauchs von T. reesei erzeugten E. coli Phytase Sequenz wurde synthetisiert und in das Plasmid pUC18 kloniert. Das erhaltene Plasmid pPhy2005 wurde durch Sequenzierung bestätigt.The fusion of the A. niger var. Awamori acid phosphatase sequence and the E. coli phytase sequence generated using codon usage of T. reesei was synthesized and cloned into the plasmid pUC18. The resulting plasmid pPhy2005 was confirmed by sequencing.
Das Plasmid ist in
Zur Konstruktion des Plasmids pAB-Phy2005 wurde die Sequenz der Fusion Phosphatase-Phytase aus dem Plasmid pPhy2005 mit AvrII und Pacl geschnitten und in die Spel- und Pacl-Spaltstellen nach dem T. reesei-cbhl-Promotor in dem Plasmid pAB489 inseriert. Das entstandene Plasmid hat die Bezeichnung pAB-Phy2005 (
Die aus dem isolierten Expressionskassette pAB-Phy2005 enthält das folgende genetische Material:
- cbhl (Cellobiohydrolase I) Promotor: Das 2,2 kb EcoRI/SacII-Fragment, das den cbhl-Promotor enthält, stammt von Trichoderma reesei QM6a. Der PromotorBereich funktioniert auch als homologe DNA (zusammen mit dem cbhl 3'-Fragment; siehe nachstehend), um die Integration der transformierenden DNA in den cbhl Lokus zu steuern.
- cbhl (cellobiohydrolase I) promoter: The 2.2 kb EcoRI / SacII fragment containing the cbhl promoter is from Trichoderma reesei QM6a. The promoter region also functions as a homologous DNA (along with the cbhl 3 'fragment, see below) to direct integration of the transforming DNA into the cbhl locus.
Saure Phosphatase-Genfragment: Das saure Phosphatase-Genfragment mit seiner Signalsequenz steht direkt unter der Kontrolle des cbhl Promotors. Die 16 Basenpaare (CCGCGGACTGCGCATC atg) stromaufwärts des Startcodons, die zu dem T. reesei-Promotor (
E. coli-Phytase-Gen: Das synthetische E. coli-Phytase-Gen ist direkt am 3'-Ende der sauren Phosphatase-Sequenz zu einem offenem Leseraster fusioniert. E. coli phytase gene: The synthetic E. coli phytase gene is fused directly to the 3 'end of the acid phosphatase sequence into an open reading frame.
cbhI-Terminator: Das 0,75 kb lange BamHI/StuI-Fragment, das den cbhl-Terminator enthält, wurde im Anschluss an die E. coli-Phytase hinzugefügt, um die Termination der Transkription zu gewährleisten.cbhI terminator: The 0.75 kb BamHI / StuI fragment containing the cbhl terminator was added following E. coli phytase to ensure termination of transcription.
amdS-Gen: Das Gen inkl. seines Promotors und seines Terminators wurde aus Aspergillus nidulans VH1-TRSX6 isoliert und codiert für Acetamidase (
cbhl 3' Fragment: Das Fragment (1,7 kb, BamHI/EcoRI, beginnend 1,4 kb nach dem Stopcodon des Gens) wurde aus T. reesei ALKO2466 isoliert. Der Stamm ALKO2466 stammt von dem Stamm ALKO233 (
Die Konstruktion des Plasmids pAB-Phy2006 erfolgte durch folgende Schritte:The construction of the plasmid pAB-Phy2006 was carried out by the following steps:
Zur Konstruktion des Plasmids pPhy2006 wurde das 87 bp lange ap-Genfragment, das für die zum C-Terminus des ap-Gens der sauren Phosphatase aus A. niger var. awamori gehörenden 29 Aminosäuren codiert, direkt an die für die letzte Aminosäure (Leucin) der E. coli Phytase codierende DNA Sequenz in dem Plasmid pPhy2005 fusioniert. Dadurch entsteht ein offenes Leseraster von 486 Aminosäuren.For the construction of the plasmid pPhy2006, the 87 bp ap gene fragment coding for the 29 amino acids belonging to the C-terminus of the ap gene of the A. niger var. Awamori acid phosphatase was directly linked to that for the last amino acid (leucine). the E. coli phytase encoding DNA sequence in the plasmid pPhy2005 fused. This creates an open reading frame of 486 amino acids.
Die Fusion Phy2006 aus der A. niger var. awamori saure Phosphatase Sequenz und der unter Verwendung des Codon-Gebrauchs von T. reesei erzeugten E. coli Phytase Sequenz wurde synthetisiert und in das Plasmid pUC18 kloniert. Die in dem erhaltene Plasmid pPhy2006 enthaltene neue Sequenz wurde durch Sequenzierung bestätigt.The fusion Phy2006 from the A. niger var. Awamori acid phosphatase sequence and the E. coli phytase sequence generated using the codon usage of T. reesei was synthesized and cloned into the plasmid pUC18. The new sequence contained in the obtained plasmid pPhy2006 was confirmed by sequencing.
Das Plasmid ist in
Die Konstruktion sowie die Klonierung des Plasmids pAB-Phy2006 ist identisch mit der in Beispiel 7 beschriebenen Herstellung des Plasmids pAB-Phy2005.The construction and the cloning of the plasmid pAB-Phy2006 is identical to the preparation of the plasmid pAB-Phy2005 described in Example 7.
Die Sequenz der Phytasevariante pAB-Phy2006 (
T. reesei RH 3780d wurde mit den aus den Plasmiden pAB489-PhyM3, pAB490-PhyM2, pAB490-PhyM7, pAB489-PhyM9, pAB600-PhyM10, pAB-Phy2005 und pAB-Phy2006 isolierten linearisierten Expressionskassetten separat transformiert. Die zur Transformation und zur Handhabung von T. reesei verwendeten Techniken waren die gemäß
Transformanten, die die Expressionskassetten aus den Beispielen 2 bis 8 tragen, bzw. die Transformanten mit dem Plasmid pKDa2 (M1) und pKDa4 (Wildtyp Aminosäuresequenz E. coli-Phytase nach Dassa) aus
Die Transformanten aus Beispiel 10 A) wurden in dem Medium aus Beispiel 10 A) in 30-1-Bioreaktoren bei einem pH-Wert von pH 3,2 ± 0,2, 200 bis 400 upm und 0,5 vvm Belüftungsrate gezüchtet. Am Ende der 6-tägigen Fermentation wurde die Biomasse durch Filtration abgetrennt und der klare Kulturüberstand mittels Ultrafiltration (30 kDa cut-off Membran) um den Faktor 6 eingeengt. Das Konzentrat wurde steril filtriert und dann für die nachfolgende Herstellung von Enzymgranulaten verwendet.The transformants of Example 10 A) were grown in the medium of Example 10 A) in 30-1 bioreactors at a pH of 3.2 ± 0.2, 200 to 400 rpm and 0.5 vvm aeration rate. At the end of the 6-day fermentation, the biomass was separated by filtration and the clear culture supernatant was concentrated by a factor of 6 by means of ultrafiltration (30 kDa cut-off membrane). The concentrate was sterile filtered and then used for the subsequent production of enzyme granules.
Die Granulate wurden aus den UF-Konzentraten durch Trocknung in einem Sprühgranulator vom Typ ProCell5, Firma Glatt Systemtechnik GmbH, Dresden, Deutschland, hergestellt. Dazu wurde der pH-Wert der UF-Konzentrate auf pH 5,2 eingestellt und vor der Trocknung 50% (w/w) Magermilchpulver mit 30% Laktosegehalt und 9,2% (w/w) Ca-Propionat, beide Zugaben bezogen auf den Gesamtproteingehalt des UF-Konzentrates, zugesetzt. Das Gemisch wurde durch eine 1,2 mm ∅ Zweistoffdüse mit einem von 1,2 auf 2,2 bar ansteigenden Luftdruck bei 80°-90°C Zulufttemperatur getrocknet. Die Aktivitätsausbeuten bei der Trocknung lagen im Bereich 89 bis 99%. Die so hergestellten Granulate wurden für den Versuch in Beispiel 12 verwendet.The granules were prepared from the UF concentrates by drying in a spray granulator of the type ProCell5, Glatt Systemtechnik GmbH, Dresden, Germany. For this purpose, the pH of the UF concentrates was adjusted to pH 5.2 and prior to drying 50% (w / w) skimmed milk powder with 30% lactose content and 9.2% (w / w) Ca propionate, both additions based on the Total protein content of the UF concentrate, added. The mixture was dried through a 1.2 mm ∅ two-component nozzle with a rising from 1.2 to 2.2 bar air pressure at 80 ° -90 ° C inlet air temperature. The activity yields in the drying were in the range of 89 to 99%. The granules thus prepared were used for the experiment in Example 12.
Die Proben aus Beispiel 10A) wurden mittels einer PD-10-Säule (Pharmacia) in 100 mM Natriumacetat Puffer, pH 5,2 umgepuffert. Dazu wurden 1,5 ml Probe aufgetragen und mit 3,5 ml Puffer nach Anleitung des Herstellers eluiert. Alle DSC-Versuche wurden auf einem VP-DSC-Gerät (MicroCal Inc., Northampton, MA, USA) durchgeführt und die Daten wurden auf einen PC übertragen um dort mit Hilfe der Software Origin v7.0383 (OriginLab Corp. Northampton, MA, USA) ausgewertet zu werden.The samples of Example 10A) were buffered by means of a PD-10 column (Pharmacia) in 100 mM sodium acetate buffer, pH 5.2. For this purpose, 1.5 ml of sample were applied and eluted with 3.5 ml of buffer according to the manufacturer's instructions. All DSC experiments were performed on a VP-DSC instrument (MicroCal Inc., Northampton, MA, USA) and the data was transferred to a PC using the Origin v7.0383 software (OriginLab Corp. Northampton, MA). USA).
Die Proben wurden vor der Messung 20 min lang in einem ThermoVac2 (MicroCal Inc., Northampton, MA; USA) entgast und auf 25°C in einem Wasserbad temperiert.The samples were degassed for 20 minutes in a ThermoVac2 (MicroCal Inc., Northampton, MA; USA) and tempered to 25 ° C in a water bath prior to measurement.
Die Messungen erfolgten mit folgenden Parametern:
- A) Konstante Geräteparameter:
- Zellvolumen: 0,51231 ml
- Referenz Heizwiderstand: 1009,1 ohms
- Zellheizwiderstand: 1002,7 ohms
- Adiabatische Rate: 0,88181
- Delta T read: 3,676
- B) VP-DSC scan Parameter waren wie folgt:
- Scanrate (aufwärts und abwärts): 1°C min-1
- Temperatur vor dem Scanzyklus: 25°C
- Äquilibrierzeit vor dem Zyklus: 15 min
- Äquilibrierzeit nach dem Zyklus: 0 min
- Filter: 10 s
- Feedback: keines
- Starttemperatur: 25°C
- Endtemperatur: 105°C
- Zelldruck: 26,1 bis 28,7 psi
- A) Constant device parameters:
- Cell volume: 0.51231 ml
- Reference heating resistor: 1009.1 ohms
- Cell heating resistor: 1002.7 ohms
- Adiabatic rate: 0.88181
- Delta T read: 3,676
- B) VP-DSC scan parameters were as follows:
- Scan rate (up and down): 1 ° C min -1
- Temperature before the scan cycle: 25 ° C
- Equilibration time before the cycle: 15 min
- Equilibration time after the cycle: 0 min
- Filter: 10 s
- Feedback: none
- Starting temperature: 25 ° C
- Final temperature: 105 ° C
- Cell pressure: 26.1 to 28.7 psi
Die nachstehende Tabelle 3 zeigt die Schmelztemperatur Tm (in Grad Celsius) und die Verschiebung der Schmelztemperatur ΔT (in Grad Celsius) der mutanten E. coli-Phytasen, die nach den oben stehenden Beispielen hergestellt wurden, im Vergleich zur Wildtyp-E. coli Phytase (Dassa et al., 1990) hergestellt mit dem gleichen Wirtssystem wie die mutanten E. coli Phytasevarianten und einer E. coli-Phytase mit einer nicht-erfindungsgemäßen Mutation (M1), ebenfalls hergestellt mit dem gleichen Wirtssystem wie die erfindungsgemäßen mutanten E. coli Phytase-Varianten.
Die Ergebnisse zeigen, dass bei allen erfindungsgemäßen Mutanten eine Erhöhung der Schmelztemperatur des Proteins eingetreten ist, was sich in einer positiven Verschiebung der Schmelzpunktstemperatur zeigt. Die Erhöhung der Schmelzpunkttemperatur ist gleichbedeutend mit einer Erhöhung der Temperaturstabilität. Die höchste Verbesserung der Thermostabilität zeigte dabei die Doppelmutation M2 mit ΔT = +1,11°C. Im Gegensatz dazu zeigte die in
Auch die Verlängerungen am N- oder N- und C-terminalen Ende zeigten Verbesserungen der Thermostabilität. Diese liegt in der Größenordnung wie die der Punktmutation bei M3.Also, extensions at the N- or N- and C-terminal ends showed improvements in thermostability. This is on the same order as that of the point mutation at M3.
Enzymgranulate hergestellt gemäß Beispiel 10B) wurden mit Weizenmehl, Typ 550, vorgemischt, um 300 g einer enzymhaltigen Vormischung zu erhalten. Diese Vormischung wurde am Biotechnologischen Institut des Research Institutes for Food and Molecular Biotechnology, Kolding, Dänemark, mit 15 kg Tierfutter vermischt um eine optimale Verdünnung für die Einmischung in 285 kg Tierfutter und eine gut bestimmbare Enzymaktivität in dem pelletierten Material zu gewährleisten. Die eingesetzte Menge an Enzymgranulat führte zu einer Phytaseaktivität von ca. 3 bis 6 Enzymeinheiten pro Gramm Tierfutter in dem Gemisch vor dem Testprozess. Die 300 kg Tierfutter die in der Pilot-Pelletieranlage behandelt wurden, haben die Zusammensetzung von Tabelle 4.
Horizontalmischer mit 700 I Volumen, 48 upm. Die Menge, die gemischt wird, liegt im Bereich 80 bis 300 kg je h.Horizontal mixer with 700 l volume, 48 upm. The amount that is mixed is in the range 80 to 300 kg per h.
An den Mischer angeschlossen ist eine Horizontalförderschnecke zum Entleeren des Mischers, deren Fördergeschwindigkeit an den nachfolgenden Schritt angepasst ist.Connected to the mixer is a horizontal screw conveyor for emptying the mixer, whose conveying speed is adapted to the subsequent step.
Anschließend an die Mischung im Horizontalmischer erfolgt eine thermische Behandlung in einem Kahl Kaskadenmischer (Konditionierer) mit 130 cm Länge, 30 cm Durchmesser, 155 upm und 37 Kammern. Durchsatz 300 kg je h. Futtermittel und Enzyme haben ca. 30 s Kontakt mit Nassdampf und werden dabei auf eine Temperatur von 70° bis 95°C erwärmt. Der Dampf, der durch einen Dan-Stoker-Hochdruckkessel bereitgestellt wird, strömt mit 2 bar Überdruck durch ein Druckregulationsventil in den Kaskadenmischer. Das Ventil regelt die Menge Dampf, die in den Kaskadenmischer einströmt und dort zur Erwärmung des Futtermittels (einschl. des Enzyms) führt.Subsequent to the mixture in the horizontal mixer is a thermal treatment in a Kahl Kaskadenmischer (conditioner) with 130 cm in length, 30 cm in diameter, 155 upm and 37 chambers. Throughput 300 kg per h. Feed and enzymes are in contact with wet steam for approx. 30 s and are heated to a temperature of 70 ° to 95 ° C. The steam that goes through a Dan Stoker high-pressure boiler is provided, flows with 2 bar overpressure through a pressure regulating valve in the cascade mixer. The valve controls the amount of steam that flows into the cascade mixer, where it warms the feed (including the enzyme).
Das konditionierte Material wird durch eine Simon Heesen Presse pelletiert und anschließend in einer mit 1500 m3 h-1 durchströmten perforierten Box durch Luft gekühlt. Die Pelletierpresse läuft mit 500 upm bei einer Leistungsaufnahme von 7,5 kW und erzeugt dabei Pellets von 3 x 20 mm.The conditioned material is pelleted by a Simon Heesen press and then air cooled in a perforated box through which the flow passes at 1500 m 3 h -1 . The pelleting press runs at 500 rpm at a power consumption of 7.5 kW, producing pellets of 3 x 20 mm.
Die Behandlung im Kaskadenmischer bewirkte eine thermische Belastung des im pelletierten Material enthaltenen Enzyms. Anschließend an die Herstellung der Pellets wurde die Wiederfindung der Phytaseaktivität im pelletierten Tierfutter bestimmt. In der nachstehenden Tabelle 5 ist die Wiederfindung der Phytaseaktivität im pelletierten Tierfutter in Abhängigkeit von der Temperatur bei der Behandlung angegeben.
Die Ergebnisse zeigen, dass die Veränderung der Thermostabilität, die bei den Messungen in Flüssigkeit mittels DSC gefunden wurde, auch in den "trockenen" Pelletierversuchen wiedergefunden wurde. Die Verbesserung der Thermostabilität gegenüber dem Wildtyp-Enzym ist dabei nicht bei allen Mutanten über den gesamten gemessenen Temperaturbereich konstant. M2 zeigte im DSC-Versuch die höchste Temperaturstabilität. Auch im Pelletierversuch wurde die beste Stabilisierung durch Mutation in einer isolierten Region durch die Doppelmutation M2 erzielt, die eine ionische Brücke in Helix D der E. coli Phytase einfügt. Die nicht erfinderische Mutante M1 zeigt wie im DSC Versuch eine Verschlechterung der Thermostabilität im Vergleich zum Wildtyp-Enzym exprimiert in Trichoderma reesei. Die ebenfalls für sich alleine nicht erfindungsgemäße Mutation M3 zeigt ähnliche Thermostabilitätseigenschaften wie das Wildtyp-Enyzm. Die Variante Phy2005, die nur die N-terminale Verlängerung durch den korrespondierenden Teil der sauren Phosphatase aus A. niger var. awamori trägt, ist nicht so stabil bei höheren Temperaturen wie Phy2006, die sowohl die N- wie auch die C-terminale Verlängerung enthält. Dies deutet darauf hin, dass diese Verlängerungen sehr wohl zu einer Zusammenlagerung von E. coli Phytase Molekülen analog zu der Dimerisierung der sauren Phosphatase unter normalen Bedingungen beitragen können und damit die Thermostabilität verbessern.The results show that the change in thermostability found in liquid measurements by DSC was also found in the "dry" pelleting experiments. The improvement in thermostability compared to the wild-type enzyme is not constant for all mutants over the entire measured temperature range. M2 showed the highest temperature stability in the DSC experiment. In the pelleting trial, the best stabilization was achieved by mutation in an isolated region through the double mutation M2, which inserts an ionic bridge into helix D of E. coli phytase. The noninventive mutant M1 shows, as in the DSC experiment, a deterioration of the thermostability in comparison to the wild-type enzyme expressed in Trichoderma reesei . The Mutation M3, which in itself is not according to the invention, shows similar thermostability properties as the wild-type enzyme. The variant Phy2005, which carries only the N-terminal extension by the corresponding part of the acid phosphatase from A. niger var. Awamori , is not as stable at higher temperatures as Phy2006, which contains both N- and C-terminal extension , This suggests that these extensions may well contribute to the aggregation of E. coli phytase molecules analogous to dimerization of the acid phosphatase under normal conditions and thus improve thermostability.
Zur Bestimmung der proteolytischen in-vitro-Stabilität wurden Enzymproben aus Beispiel 10A) verwendet, d.h. Proben der Transformanten M1 (der nicht erfindungsgemäßen Mutation), M2, M3, M7, Phy2005 und Phy2006 hergestellt mit den Transformaten aus Tabelle 2.To determine in vitro proteolytic stability, enzyme samples from Example 10A) were used, i. Samples of the transformants M1 (the mutation not according to the invention), M2, M3, M7, Phy2005 and Phy2006 prepared with the transformants from Table 2.
20 ml einer Pepsin-Lösung (Merck 7190), die 20 Proteaseeinheiten pro ml in Glycin-HCl-Puffer (pH 2,5) enthält, wobei die Aktivität nach Angabe des Herstellers auf Hämoglobin, pH 1,6, 25°C bezogen ist, wurde bei 40°C temperiert. 10 ml einer Phytase-Lösung (1:100 verdünnt) wurde zugegeben und die Lösung mit Puffer auf 50 ml aufgefüllt und anschließend bei 40°C, pH 2,5 für 30 min inkubiert. Die Reaktion wurde durch Eintauchen in ein Eis/Wasser-Bad gestoppt und der pH-Wert wurde sofort auf pH 5 mit Natronlauge angehoben und die Lösung dabei auf 100 ml verdünnt. Die Phytaseaktivität wurde dann nach Beispiel 1 gemessen.20 ml of a pepsin solution (Merck 7190) containing 20 protease units per ml in glycine-HCl buffer (pH 2.5), the activity being based on hemoglobin, pH 1.6, 25 ° C according to the manufacturer's instructions , was tempered at 40 ° C. 10 ml of a phytase solution (diluted 1: 100) was added and the solution was made up to 50 ml with buffer and then incubated at 40 ° C, pH 2.5 for 30 min. The reaction was stopped by immersion in an ice / water bath and the pH was immediately raised to pH 5 with sodium hydroxide solution and the solution was diluted to 100 ml. The phytase activity was then measured according to Example 1.
10 ml der mit Pepsin behandelten Lösung wurde auf pH 7 eingestellt. 30 ml einer Pankreatin-Lösung (Merck 7130), die 30 Proteaseeinheiten (Casein, pH 8,0, 35°C), in 0,05 M Tris-HCl-Puffer (pH 7,0) enthält, wurde zugegeben, die Lösung mit Tris-HCl-Puffer, pH 7, auf 50 ml verdünnt und die Lösung in einem Wasserbad bei 40°C für 30 min inkubiert. Die Reaktion wurde durch Eintauchen in ein Eis/Wasser-Bad gestoppt und der pH-Wert wurde sofort auf pH 5 mit HCl erniedrigt und die Lösung dabei auf 100 ml verdünnt. Die Phytaseaktivität wurde dann nach Beispiel 1 gemessen. Die Ergebnisse sind in Tabelle 6 dargestellt.
Die Ergebnisse zeigen für alle erfindungsgemäßen Mutanten eine sehr hohe proteolytische Stabilität in Gegenwart von Pepsin und Pankreatin, verglichen mit der Vergleichsmutante PhyM1, unter in-vitro-Bedingungen, wie sie im Magen von Monogastriern, z. B. Schweinen, vorkommen. Die hier gezeigte hohe proteolytische Stabilität verdeutlicht, dass diese Enzymmutanten für einen Einsatz im Tierfutterbereich besonders geeignet sind. The results show for all mutants of the invention a very high proteolytic stability in the presence of pepsin and pancreatin compared to the control mutant PhyM1 under in vitro conditions as found in the stomach of monogastric animals, e.g. As pigs, happen. The high shown here Proteolytic stability makes it clear that these enzyme mutants are particularly suitable for use in the animal feed sector.
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EP07819547A EP2102334B1 (en) | 2006-11-10 | 2007-11-02 | Polypeptide having phytase activity and increased temperature resistance of the enzyme activity, and nucleotide sequence coding said polypeptide |
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ES2737885T3 (en) * | 2010-03-26 | 2020-01-16 | Novozymes As | Thermostable variants of phytase |
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CN113005137B (en) * | 2021-02-25 | 2022-10-11 | 石河子大学 | Construction method of regulatory element with dual functions of starting and stopping, dual-function element library and application |
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EP2102334B1 (en) | 2013-04-03 |
PL2102334T3 (en) | 2013-09-30 |
WO2008055625A2 (en) | 2008-05-15 |
EP2428565B1 (en) | 2013-09-25 |
DK2428565T3 (en) | 2013-12-02 |
ES2434328T3 (en) | 2013-12-16 |
CN101631855B (en) | 2014-08-06 |
US20110262619A1 (en) | 2011-10-27 |
US8420369B2 (en) | 2013-04-16 |
US9353357B2 (en) | 2016-05-31 |
DK2102334T3 (en) | 2013-04-29 |
PL2428565T3 (en) | 2014-02-28 |
ES2406758T3 (en) | 2013-06-10 |
WO2008055625A3 (en) | 2009-03-19 |
EP2102334A2 (en) | 2009-09-23 |
US20140242249A1 (en) | 2014-08-28 |
DE102006053059A1 (en) | 2008-05-15 |
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